B- AND y -DIKETONES AND y -HYDROXYKETONES AS WNT/ B -CATENIN SIGNALING PATHWAY ACTIVATORS

ABSTRACT

which activates Wnt/β-catenin signaling and thus treats or prevents diseases related to signal transduction, such as osteoporosis and osteoarthropathy; osteogenesis imperfecta, bone defects, bone fractures, periodontal disease, otosclerosis, wound healing, craniofacial defects, oncolytic bone disease, traumatic brain injuries related to the differentiation and development of the central nervous system, comprising Parkinson&#39;s disease, strokes, ischemic cerebral disease, epilepsy, Alzheimer&#39;s disease, depression, bipolar disorder, schizophrenia; eye diseases such as age related macular degeneration, diabetic macular edema or retinitis pigmentosa and diseases related to differentiation and growth of stem cell, comprising hair loss, hematopoiesis related diseases and tissue regeneration related diseases.

RELATED APPLICATIONS Cross-Reference to Related Applications

This application is a continuation application of U.S. application Ser.No. 15/855,463, filed Dec. 27, 2017, which is a continuation applicationof U.S. application Ser. No. 15/349,118, filed Nov. 11, 2016, which is acontinuation application of U.S. application Ser. No. 14/547,951, filedNov. 19, 2014, which is a continuation of U.S. application Ser. No.14/086,529, filed Nov. 21, 2013, which is a continuation application ofU.S. application Ser. No. 13/211,665, filed Aug. 17, 2011, and claimsthe benefit of U.S. Provisional Application No. 61/374,687, filed Aug.18, 2010, and U.S. Provisional Application No. 61/427,974, filed Dec.29, 2010, each of which is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates to activators of one or more proteins in the Wntpathway, including activators of one or more Wnt proteins, andcompositions comprising the same. More particularly, it concerns the useof a β-diketone, γ-diketone or γ-hydroxyketone or salts or analogsthereof, in the treatment of osteoporosis and osteoarthropathy;osteogenesis imperfecta, bone defects, bone fractures, periodontaldisease, otosclerosis, wound healing, craniofacial defects, oncolyticbone disease, traumatic brain injuries related to the differentiationand development of the central nervous system, comprising Parkinson'sdisease, strokes, ischemic cerebral disease, epilepsy, Alzheimer'sdisease, depression, bipolar disorder, schizophrenia; eye diseases suchas age related macular degeneration, diabetic macular edema or retinitispigmentosa and diseases related to differentiation and growth of stemcell, comprising hair loss, hematopoiesis related diseases and tissueregeneration related diseases.

Description of the Related Art

The Wnt/β-catenin signaling pathway is essential in many biologicalprocesses. It regulates the fate of as-yet undeveloped cells in embryoform. The Wnt/β-catenin signaling pathway is essential to stem cellself-renewal and proliferation as well as the development of stem cellsin adult organisms (e.g. skin cell, bone cell, liver cell, etc.)[Science (2002), 296(5573), 1644-1646]. The Wnt/β-catenin signalingpathway regulates development, morphology, proliferation, motility andcell fate [Annual Review of Cell and Developmental Biology (2004), 20,781-810]. The Wnt/β-catenin signaling pathway has a central role intumorigenesis and inappropriate activation of this system is observed inseveral human cancers [“Wnt Signaling in Human Cancer”, in SignalTransduction in Cancer (pp. 169-187). (2006) Springer]. Wnt/β-cateninwas first described in humans as a protein which interacts with thecytoplasmic domain of E-cadherin and with Wnt/β-catenin, anchoring thecadherin complex to the actin cytoskeleton [Science (1991), 254(5036),1359-1361]. Then, an additional role for mammalian Wnt/β-catenin wasdiscovered; namely, as the key mediator of Wnt/β-catenin messaging.

In the presence of a Wnt ligand, if not inhibited by secretedantagonists, the Wnt ligand binds a frizzled (Fzd)/low densitylipoprotein receptor related protein (LRP) complex, activating thecytoplasmic protein dishevelled (Dsh in Drosophila and Dvl invertebrates). Precisely how Dsh/Dvl is activated is not fullyunderstood, but phosphorylation by casein kinase 1 (CK1) and caseinkinase 2 (CK2) have been suggested to be partly responsible [Proceedingsof the National Academy of Sciences of the USA (1999), 96(22),12548-12552]. Dsh/Dvl then inhibits the activity of the multiproteincomplex (β-catenin-Axin-adenomatous polyposis coli (APC)-glycogensynthase kinase (GSK)-3β), which targets β-catenin by phosphorylationfor degradation by the proteasome. Dsh/Dvl is suggested to bind CK1 andthereby inhibiting priming of β-catenin and indirectly preventing GSK-3βphosphorylation of β-catenin [Genes & Development (2002), 16(9),1066-1076]. Upon Wnt stimulation, Dvl has also been shown to recruitGSK-3 binding protein (GBP) to the multiprotein complex. GBP mighttitrate GSK-3β from Axin and in this way inhibits phosphorylation ofβ-catenin. Finally, sequestration of Axin at the cell membrane by LRPhas been described [Molecular cell (2001), 7(4), 801-809]. The overallresult is accumulation of cytosolic β-catenin. Stabilized β-catenin willthen translocate into the nucleus and bind to members of the T-cellfactor (Tcf)/Lymphoid enhancing factor (Lef) family of DNA bindingproteins leading to transcription of Wnt target genes.

In the absence of a Wnt ligand, Axin recruits CK1 to the multiproteincomplex causing priming of β-catenin and initiation of the β-cateninphosphorylation cascade performed by GSK-3β. Phosphorylated 3-catenin isthen recognized by 3-transducin repeat-containing protein (β-TrCP) anddegraded by the proteasome, reducing the level of cytosolic β-catenin.

Aberrant activation of the Wnt/β-catenin pathway has led to severalphenotypes, including the development of a variety of human cancers, anddiseases leading to abnormal development and functioning of the stemcells [Oncogene (2009), 28(21), 2163-2172; Cancer Cell (2008), 14(6),471-484; American Journal of Pathology (2003), 162(5), 1495-1502].Chronic activation of the Wnt/β-catenin signaling pathway has beenimplicated in the development of a variety of human malignancies,including high bone mass syndrome, sclerosteosis, colorectal carcinomas,hepatocellular carcinomas (HCCs), ovarian, uterine, pancreaticcarcinomas, and melanomas [BioEssays (1999) 21(12), 1021-1030; Cell(2000), 103(2), 311-320; Genes Dev. (2000), 14(15), 1837-1851]. Sincethe Wnt/β-catenin pathway is involved in myriad growth and developmentprocesses, mutation of the proteins involved in the Wnt/β-catenin signaltransduction system is closely correlated with various human diseasessuch as abnormalities in development, hair follicle morphogenesis, stemcell differentiation, bone formation and cell proliferation.

Hair Loss

Hair forms in a pouch-like structure below the skin called a hairfollicle. Visible hair, for example that seen on a human scalp, isactually the hair shaft, which is keratinized, hardened tissue thatgrows from the hair follicle. In particular, the hair shaft is composedlargely of keratin, which is produced by keratinocytes.

Normal hair follicles cycle between a growth stage (anagen), adegenerative stage (catagen), and a resting stage (telogen). Scalp hairshave a relatively long life cycle: the anagen stage ranges from 2 to 6years, the catagen stage ranges from a few days to a few weeks, and thetelogen stage is approximately three months. Shorter hairs foundelsewhere on the human body have corresponding shorter anagen durations.The morphology of the hair and the hair follicle change dramaticallyover the course of the life cycle of the hair [Dermatology in GeneralMedicine (Vol. I), McGraw-Hill, Inc., 1993, pp. 290-91; Sperling, L. C.;J. Amer. Acad. Dermatology (1991), 25(1, Part 1), 1-17].

During anagen, the hair follicle is highly active metabolically. Thefollicle comprises a dermal papilla at the base of the follicle; andepidermal matrix cells surrounding the dermal papilla form the base ofthe hair shaft, which extends upwards from the papilla through the haircanal. The matrix cells are the actively growing portion of the hair.

At catagen, the matrix cells retract from the papilla, and otherdegenerative changes occur. For example, the vessels and capillariessupplying blood and nutrients to the hair follicle shrivel and stopfunctioning. A column of epithelial cells pushes the keratinizedproximal shaft of the hair upwards, and cell death occurs within thefollicle. The hair shaft is then shed from the scalp or other part ofthe body and the hair follicle enters telogen, the resting stage of thehair growth cycle.

Although hair follicle regulation and growth are not well understood,they represent dynamic processes of proliferation, differentiation, andcellular interactions during tissue morphogenesis. It is believed thathair follicles are formed only in the early stages of development andare not replaced. Thus, an increase in damaged or non-functioning hairfollicles is generally associated with hair loss.

Male or female pattern baldness requires the presence of male or femalehormones, e.g. androgens, but the cause is unknown. The extent of hairloss in any male greatly depends on the genes he inherits from hisfather, mother, or both. Hair loss begins at the temples or at the topof the head. If male pattern hair loss begins in the mid-teens,subsequent hair loss is usually fairly extensive. Male balding goes inwaves. Hair loss may begin in the early 20's, then stop, only to startagain in a few years. By the age of 20 to 30 years, 30% of men have baldspots. This continues to rise until age 50-60, when 50% of men arecompletely bald.

The rate of hair loss is affected by advancing age, the tendency to baldearly due to inherited genes, and an overabundance of the male hormonedihydrotestosterone (DHT) within the hair follicle. DHT acts on ahormone receptor within the hair follicle, and thereby slows hairproduction and produces weak, shorter hair. Sometimes DHT productioneven stops hair growth completely. Although balding men have aboveaverage amounts of DHT in their hair follicles, they usually do not haveabove average circulating testosterone levels.

Female pattern baldness is not as common as male pattern baldness, butis on the rise. It is confined to the hair predominantly at the top ofthe head and complete baldness is rare in females.

Toxic alopecia is temporary but typically lasts three to four months,and often is caused by an infectious disease. For example, toxicalopecia may occur as a result of hypothyroidism, diabetes, hormonalproblems, vitamin deficiencies, hypopituitarism, parasites, poordigestion, early stage of syphilis, vitamin A or retinoid overdoses, orother cytotoxic drugs.

Alopecia areata is a sudden hair loss in demarcated areas. It can affectany hairy area, but most frequently affects the scalp and beard. Hairloss confined to a few areas is often reversed in a few months evenwithout treatment but recurrence is a possibility. Alopecia areatausually occurs in people with no obvious skin disease or systemicdisease, but in rare cases lab tests may show anti-microsomal antibodiesto thyroglobulin, gastric parietal cells and adrenal cells.

Scarring alopecia results from inflammation and tissue destruction. Itmay be due to injuries such as burns, physical trauma, or destructionafter x-rays. In these cases, little regrowth is expected. Other causesare cutaneous lupus erythematosus, lichen planus, chronic deep bacterialor fungal infections, deep ulcers, sarcoidosis, syphilis, ortuberculosis. Slow growing tumors of the scalp are a rare cause of hairloss.

While none of these conditions is very well understood, each conditionis distressing because hair is often considered an important factor inhuman social communications and interactions.

Numerous approaches have been suggested for treating hair loss. Two ofthe most commonly used and accepted compounds for preventing hair lossare minoxidil, the active ingredient in Rogaine® and the 5α-reductaseinhibitor, finasteride, the active ingredient in Propecia®. However,cosmetic treatment of age-related hair loss in patients with topicalsolution of minoxidil or finasteride has resulted in only moderateregrowth of hair in less than 40% of such patients. Indeed, less thanten percent of the men who use Rogaine® achieve satisfactory results.Thus, there is a need in the art for more effective methods of, andcompositions for treating hair loss. Preferably, new methods andcompositions will require fewer applications of active ingredients;provide hair regrowth sooner, in more abundance, and thicker, thanpresently observed with minoxidil or finasteride treatment.

It has been found that hair follicle development and regeneration areregulated by the canonical Wnt/β-catenin signaling pathway[Investigative Dermatology (2008), 128(5), 1081-1087]. In the epidermis,hair follicle development is initiated when mesenchymal cells populatethe skin. During this process, signals emanating from the dermis induceepithelium thickening, elongation of the epithelial cells, and theformation of placodes containing Wnt-responsive cells. In response,placodes signal dermal cells to condense, thereby forming the dermalpapilla component of the hair follicle, which is also responsive to Wntsignaling. Wnt3a is secreted from hair epithelium and acts in anautocrine and paracrine fashion, and it has been demonstrated thatWnt-3a maintains anagen gene expression in dermal papilla cells andmediates hair-inductive activity in an organ culture. ThisWnt-3a-mediated hair growth might depend on the canonical Wnt/β-cateninsignaling pathway because deletion of β-catenin or the Lef1 generesulted in hair loss in mice. Therefore, activation of β-catenin by Wntcontributes to the inhibition of keratinocytes differentiation,induction of hair follicle formation, and maintenance of proliferationof neuronal progenitors.

Neurodegenerative Diseases

Neurodegenerative diseases result from deterioration of neurons or theirmyelin sheath which over time will lead to dysfunction and disabilitiesresulting from this. Adult mammalian brain has limited capacity forregeneration. This makes the repair of any injuries hazardous and,consequently, CNS traumas are devastating.

New neurons are generated from neural stem cells, in two regions of theadult mammalian central nervous system: the subventricular zone of thelateral ventricle, and the subgranular zone of the hippocampal dentategyrus [Current Opinion in Cell Biology (2001), 13, 666-672]. Signalsprovided by the microenvironment contribute to the regulation of themaintenance, proliferation and neuronal fate commitment of the localstem cells. Many of these signals and signaling pathways are unknown.

Alzheimer's disease (AD) is the most common cause of dementia thatgradually destroys neurons and affects more than 24 million peopleworldwide. It occurs mostly in older adults and patients afflicted withAD lose their ability to learn, remember, make decisions, communicateand carry out daily activities. The etiology and progression of AD isnot well understood, but is associated with amyloid beta (Aβ) plaquesand neurofibrillary tangles in the brain.

Parkinson's disease (PD) is a degenerative disorder of the centralnervous system affecting more than 6 million people worldwide and thatoften impairs the sufferer's motor skills and speech. The symptoms ofParkinson's disease result from the loss of dopamine-secreting cells inthe region of the substantia nigra (literally “black substance”). Theseneurons project to the striatum and their loss leads to alterations inthe activity of the neural circuits within the basal ganglia thatregulate movement.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative diseasethat results from the death of motor neurons. A progressive loss ofmuscle control impairs the individual's capacity for independentfunction. ALS strikes the cells in the brain and spinal cord (motorneurons), which send signals to move muscles. In some cases, a mutationin the SOD1 gene results in a dysfunctional protein, the superoxidedismutase protein (called SOD1), which normally “cleans” up toxicparticles inside a cell. When SOD1 is mutated, toxic particlesaccumulate inside motor neurons causing them to malfunction. But thismutation only explains a few percent of cases of ALS. The primary causeof ALS, which afflicts about 350,000 adults worldwide, is unknown.

Stroke and traumatic brain injury can also cause neuronal loss and leadto cognitive decline. Stroke can be classified into two majorcategories: ischemic and hemorrhagic. Ischemia is due to interruption ofthe blood supply, while hemorrhage is due to rupture of a blood vesselor an abnormal vascular structure. Stroke can cause permanentneurological damage, complications and death if not promptly diagnosedand treated. It is the third leading cause of death and the leadingcause of adult disability in the United States and Europe.

Frontotemporal Dementia (FTD) accounts for 18% of dementias in peopleunder 65 years old. It frequently manifests itself as a behavioraldisturbance, and can progress to impair an individual's capacity forindependent thought and function. Recent studies have uncovered geneticfactors that contribute to this dementia; however no treatment yetexists to block the brain deterioration it causes.

Wnt/β-catenin signal transduction system plays a crucial role in thedifferentiation and development of nerve cells for the central nervoussystem, suggesting a relationship between Wnt/β-catenin proteins and theincidence of various diseases of the central nervous system, includingneurodegenerative diseases [Nature (2005), 437(7063), 1370-1375].Particularly, it is also found that Wnt/β-catenin signaling is relatedto diseases resulting from the abnormality of nerve cells, such as braindamage, Parkinson's disease, Amyotrophic Lateral Sclerosis (Lou Gehrig'sdisease), stroke, epilepsy, Alzheimer's disease (AD), depression,bipolar disorder, and schizophrenia.

Alzheimer's disease is the most common age-related neurodegenerativedisorder. In fact, a relationship between amyloid-β-peptide (Aβ)-inducedneurotoxicity and a decrease in the cytoplasmic levels of β-catenin hasbeen observed. Apparently Aβ binds to the extracellular cysteine-richdomain of the Frizzled receptor (Fz) inhibiting Wnt/β-catenin signaling.Cross-talk with other signaling cascades that regulate Wnt/β-cateninsignaling, including the activation of M1 muscarinic receptor and PKC,the use of Ibuprofen-ChE bi-functional compounds, PPAR α, γ agonists,nicotine and some antioxidants, results in neuroprotection against Aβ.These studies indicate that a sustained loss of Wnt signaling functionmay be involved in the Aβ-dependent neurodegeneration observed inAlzheimer's brain. Thus, the activation of the Wnt/β-catenin signalingpathway could be proposed as a therapeutic target for the treatment ofAD.

Eye Diseases

Age related macular degeneration (AMD) is a medical condition whichusually affects older adults that results in a loss of vision in thecenter of the visual field (the macula) because of damage to the retina.It occurs in “dry” and “wet” forms. It is a major cause of visualimpairment in older adults (>50 years). The inner layer of the eye isthe retina, which contains nerves that communicate sight, and behind theretina is the choroid, which contains the blood supply to the macula(the central part of the retina). In the dry (nonexudative) form,cellular debris called drusen accumulate between the retina and thechoroid, and the retina can become detached. In the wet (exudative)form, which is more severe, blood vessels grow up from the choroidbehind the retina, and the retina can also become detached. It can betreated with laser coagulation, and with medication that stops andsometimes reverses the growth of blood vessels.

Diabetic retinopathy is retinopathy (damage to the retina) caused bycomplications of diabetes mellitus, which can eventually lead toblindness. It is an ocular manifestation of systemic disease whichaffects up to 80% of all patients who have had diabetes for 10 years ormore. As new blood vessels form at the back of the eye as a part ofproliferative diabetic retinopathy (PDR), they can bleed (hemorrhage)and blur vision. Some people develop a condition called macular edema.It occurs when the damaged blood vessels leak fluid and lipids onto themacula, the part of the retina that lets us see detail. As the diseaseprogresses, severe nonproliferative diabetic retinopathy enters anadvanced, or proliferative, stage. The lack of oxygen in the retinacauses fragile, new, blood vessels to grow along the retina and in theclear, gel-like vitreous humour that fills the inside of the eye.Without timely treatment, these new blood vessels can bleed, cloudvision, and destroy the retina.

Retinitis pigmentosa (RP) is a group of genetic eye conditions. In theprogression of symptoms for RP, night blindness generally precedestunnel vision by years or even decades. Many people with RP do notbecome legally blind until their 40s or 50s and retain some sight alltheir lives [American Journal of Ophthalmology (2003), 136(4), 678-68].Others go completely blind from RP, in some cases as early as childhood.Progression of RP is different in each case. RP is a type of progressiveretinal dystrophy, a group of inherited disorders in which abnormalitiesof the photoreceptors (rods and cones) or the retinal pigment epithelium(RPE) of the retina lead to progressive visual loss. Affectedindividuals first experience defective dark adaptation or nyctalopia(night blindness), followed by reduction of the peripheral visual field(known as tunnel vision) and, sometimes, loss of central vision late inthe course of the disease.

Müller glia, or Müller cells, are glial cells found in the vertebrateretina, which normally serve the functions of any normal glial cells.However, following injury to the retina, it has been seen that Müllerglia undergo dedifferentiation into multipotent progenitor cells. Atthis point, the progenitor cell can divide and differentiate into anumber of retinal cell types, including photoreceptors, that may havebeen damaged during injury. Additionally, recently published researchhas shown that Müller cells act as a light collector in the mammalianeye, analogous to a fiber optic plate, funneling light to the rod andcone cells.

Multipotent retinal progenitors must solve two fundamental problems.First, they must initially expand their numbers but later limit theirproliferation so that the right number of differentiated cells isproduced at the appropriate developmental time. Second, the distinctprocesses of division and differentiation must be coordinated so thatdifferentiation can be initiated when cells stop dividing [CurrentOpinion in Genetics & Development (1997), 7(5), 651-658; Nature ReviewsNeuroscience (2001), (2), 109-118]. Wnt promotes cell proliferation inmultiple tissues [Cell and Tissue Research (2008), 331(1), 193-210], inparticular in the developing retina [Stem Cells (2008), 26(8),2063-2074; Development (2003), 130(3), 587-598; Development (2005),132(12), 2759-2770; Development (2005), 132(13), 3027-3043]. The SoxB1family of genes (Sox1-3) may be key effectors of Wnt/β-catenin signalingin the developing nervous system [Development (2006), 133(22),4451-4461; Neuron (2005), 46(1), 23-36]. During neurogenesis, Sox2antagonizes proneural genes and can maintain progenitors [NatureNeuroscience (2003), (6), 1162-1168; Neuron (2003), 39(5), 749-765]. Inthe frog retina, Wnt/β-catenin signaling through Fz5 is necessary forSox2 expression, which is required for proneural gene expression and thetransition from progenitors to neurons [Neuron (2005), 46(1), 23-36]. Itwas discovered that these factors are core components of a conservedhierarchical cascade and propose that they form a powerful directionalnetwork that drives cells from a proliferative, undifferentiated stateto a nonproliferative, differentiated neuronal or glial fate[Development (2009), 136(19), 3289-3299].

Regeneration in the mammalian CNS is severely limited. Unlike in thechick, current models hold that retinal neurons are never regenerated.It has been demonstrated that, in the adult mammalian retina, Müllerglia dedifferentiate and produce retinal cells, includingphotoreceptors, after acute neurotoxic injury in vivo. However, thenumber of newly generated retinal neurons is very limited. It has beendemonstrated that Wnt/β-catenin signaling promotes proliferation ofMüller glia-derived retinal progenitors and neural regeneration afterdamage or during degeneration. Wnt3a treatment increases proliferationof dedifferentiated Müller glia>20-fold in the photoreceptor-damagedretina. It has also been shown that in the degenerating retina, Wnt3aincreased cell proliferation, and treatment with RA or VPA promoted thedifferentiation of these cells into rhodopsin-positive photoreceptorcells [Journal of Neuroscience (2007), 27(15), 4210-4219].

Therefore, we propose that modulating the Wnt/β-catenin pathway is onepossible therapeutic strategy to enhance replacement of lost neurons bygenerating cells derived from endogenous neuronal progenitors.

Bone Formation

Canonical Wnt/β-catenin signaling has been demonstrated to increase boneformation, and Wnt pathway components are being pursued as potentialdrug targets for osteoporosis and other metabolic bone diseases [Bone(2009), 44(6), 1063-1068]. In modern times, bone diseases are increasingdue to socio-environmental and genetic factors, particularly due toincrease of population of elderly persons. Generally, bone diseasesoccur and develop without special symptoms, and rapidly worsen with age.Although many drugs have been developed for the treatment of bonediseases thus far, most of them mainly aim to alleviate pain or toretard the decrease of bone density. They are not effective as acurative medication which aims for increasing the bone density ofpatients who suffer from osteoporosis. Some other drugs are usually inthe form of injections and are reported to produce side effects uponlong-term administration thereof.

Signaling through the Wnt/β-catenin pathway can increase bone massthrough a number of mechanisms, including renewal of stem cells,stimulation of preosteoblast replication, induction ofosteoblastogenesis, and inhibition of osteoblast and osteocyteapoptosis. One molecular mechanism is through the stimulation of the Wntpathway by Wnt-3a interaction of its receptors LRP5 and Fzd [Journal ofMedicinal Chemistry (2009), 52(22), 6962-6965]. Bone forming osteoblastsexpress the proteins LRP5 and Fzd on the surface membrane, which serveas co-receptors for the soluble peptide agonist Wnt-3a. Once stimulatedwith Wnt-3a, internal concentrations of free 3-catenin rise and enterthe nucleus and recruit T-cell factor (TCF). Transcriptional eventsfollow and result in the production of additional anabolic geneproducts. An additional soluble extracellular protein, Dkk-1,antagonizes this process by simultaneously binding to the cell surfacereceptors Kr2 and LRP5, effectively inhibiting Wnt-3a binding to LRP5.In addition, the Kr2/LRP5/Dkk-1 complex undergoes endocytosis to removeLRP5 from the cell membrane, thereby nullifying its function.Loss-of-function mutations of secreted Wnt antagonists like Dkk-1,SOST/sclerostin and secreted frizzled-related protein (sFRP)-1 result inincreased bone formation due to changes in a variety of osteoblastparameters like proliferation, differentiation, recruitment/longevityand function [Journal of Bone and Mineral Research (2009), 21(6),934-945], while deletion of the 0-catenin-activated transcription factorTCF-1 causes osteopenia that arises from a reduction in osteoprotegerinexpression by the osteoblast [Developmental Cell (2005), 8(5), 751-764].

Intestinal Diseases

The adult intestinal epithelium is characterized by continuousreplacement of epithelial cells through a stereotyped cycle of celldivision, differentiation, migration and exfoliation occurring during a5-7 day crypt/villus transit time. The putative growth factorsregulating proliferation within the adult intestinal stem cell nichehave not yet been identified, although studies have implicated thecell-intrinsic action of 3-catenin/Lef/Tcf signaling within theproliferative crypt compartment.

A number of pathological conditions affect the cells of the intestines.Inflammatory bowel disease (IBD) can involve either or both the smalland large bowel. Crohn's disease and ulcerative colitis are thebest-known forms of IBD, and both fall into the category of “idiopathic”inflammatory bowel disease because the etiology for them is unknown.“Active” IBD is characterized by acute inflammation. “Chronic” IBD ischaracterized by architectural changes of crypt distortion and scarring.Crypt abscesses can occur in many forms of IBD.

Ulcerative colitis (UC) involves the colon as a diffuse mucosal diseasewith distal predominance. The rectum is virtually always involved, andadditional portions of colon may be involved extending proximally fromthe rectum in a continuous pattern. The etiology for UC is unknown.Patients with prolonged UC are at increased risk for developing coloncancer.

Patients with UC are also at risk for development of liver diseasesincluding sclerosing cholangitis and bile duct carcinoma.

Crohn's disease can involve any part of the GI tract, but mostfrequently involves the distal small bowel and colon. Inflammation istypically transmural and can produce anything from a small ulcer over alymphoid follicle (aphthoid ulcer) to a deep fissuring ulcer totransmural scarring and chronic inflammation. One third of cases havegranulomas, and extracolonic sites such as lymph nodes, liver, andjoints may also have granulomas. The transmural inflammation leads tothe development of fistulas between loops of bowel and other structures.Inflammation is typically segmental with involved bowel separating areasof involved bowel. The etiology is unknown, though infectious andimmunologic mechanisms have been proposed.

Gluten, a common dietary protein present in wheat, barley and rye causesa disease called Celiac disease in sensitive individuals. Ingestion ofsuch proteins by sensitive individuals produces flattening of thenormally luxurious, rug-like, epithelial lining of the small intestine.

Other clinical symptoms of Celiac disease include fatigue, chronicdiarrhea, malabsorption of nutrients, weight loss, abdominal distension,anemia, as well as—a substantially enhanced risk for the development ofosteoporosis and intestinal malignancies such as lymphoma and carcinoma.Celiac disease is generally considered to be an autoimmune disease andthe antibodies found in the serum of the patients support the theorythat the disease is immunological in nature.

Transgenic mice that have a knock-out of the Tcf locus show a loss ofproliferative stem cell compartments in the small intestine during lateembryogenesis [Oncogene (2006) 25(57), 7512-7521]. However, the knockoutis lethal, and so has not been studied in adults. In chimeric transgenicmice that allow analysis of adults, expression of constitutively activeNH₂-truncated β-catenin stimulated proliferation in small intestinecrypts, although either NH₂-truncated β-catenin or Lef-1/□-cateninfusions induced increased crypt apoptosis as well [The Journal of CellBiology (1998), 141(3), 765-777; The Journal of Biological Chemistry(2002), 277(18), 15843-15850]. Because diverse factors regulateβ-catenin/Lef/Tcf-dependent transcription, including non-Frizzled GPCRsand PTEN/PI-3-kinase, the cause of intestinal stem cell defect is notknown. Genes expressed in the gastrointestinal tract that are controlledby Wnt/β-catenin include CD44, and EphB2.

Regenerative Medicine

Due to the remarkable advances made in the field of medicine in recentyears, opportunities for saving lives are continuing to increase in thearea of living donor transplant techniques for tissues and organs.However, there are limitations on treatment dependent upon living donortransplants due to such factors as a shortage of transplant donors andthe occurrence of rejection. If it were possible to regenerate a tissueor organ that has been lost due to surgical treatment or an unforeseenaccident, then it would be possible to considerably improve the qualityof life for patients. In addition, regenerative medicine also makes itpossible to resolve the problems confronting living donor transplants.From this viewpoint, the degree of expectations being placed onregenerative medicine is high.

Technologies in which regenerative medicine has been successful areprimarily related to comparatively simple tissue in terms of morphologyor function in the manner of artificial skin, artificial bone andartificial teeth. Reconstructed artificial skin and artificial bone isincorporated into cells enabling the providing of signals required fortissue construction. However, there have been limitations on therepertoire of differentiation of artificial skin and artificial bone byregenerative medicine techniques. For example, although allogeneickeratinocytes or skin fibroblasts and the like differentiate intostructures in the form of the epidermis, are incorporated by surroundingorgans to eventually have a horny layer or basal layer having barrierproperties, there has been reported to be no derivation of secondaryderivatives such as hair follicles, sebaceous glands or sweat glands.

Body tissue normally contains both cells that are able to self-replicateand possess stem cell properties for maintaining tissue homeostasis bysending signals to differentiated cells or supplying differentiatedcells, and cells having properties of somatic cells that have alreadydifferentiated that receive various signals or commands from such cells,and is able to function through interaction between both of these typesof cells. In the case of vertebrates, for example, interaction betweenmesenchymal cells and epithelial cells is essential for nearly alltissue and organ formation. In the case of hair follicles, mesenchymalcells in the form of hair papilla cells are responsible for stemcell-like properties, while epithelial cells in the form ofkeratinocytes are equivalent to cells having somatic cell-likeproperties in their ability to differentiate into hair shafts (hairitself).

The difficulty encountered when forming organs by regenerative medicinelies in reaching a state of coexistence between cells having stemcell-like properties maintained in an undifferentiated state and cellsthat have already differentiated as in actual body tissue. In the priorart, even if epithelial cells and mesenchymal cells were able to beco-cultured, they either both ended up differentiating or bothmaintained an undifferentiated state, thereby preventing thereproduction of the coexistence of undifferentiated cells anddifferentiated cells so as to mimic actual body tissue.

Guiding multipotent cells into distinct lineages and controlling theirexpansion remain fundamental challenges in developmental and stem cellbiol. Members of the Wnt pathway control many pivotal embryonic events,including self-renewal or expansion of progenitor cells.

Published observations suggest that canonical Wnt signals play distinctroles during discrete developmental windows, first positively regulatingmesoderm commitment and then possibly playing a negative role in theinitial induction of cardiac progenitors [Genes & Development (2001),15(3), 316-327; Ibid., 304-315; Proc Natl Acad Sci USA. (2006), 103(52),19812-19817; Development (Cambridge, UK) (2006), 133(19), 3787-3796].The loss- and gain-of-function studies of canonical Wnt signaling in aspatiotemporally restricted manner described here provide compellingevidence that Wnt/β-catenin signaling is required in a cell autonomousfashion for the expansion and development of precardiac mesoderm andcardiac mesoderm in mouse. Thus, narrow developmental windows may existduring which canonical Wnt signaling sequentially inhibits then promotescardiac development. Thus it was shown that canonical Wnt signaling canbe manipulated to regulate expansion and differentiation of progenitorcells.

In contrast to progenitor cells, however, stem cells are far lessspecific. The most important difference between stem cells andprogenitor cells is that stem cells can replicate indefinitely, whereasprogenitor cells can only divide a limited number of times. The termadult stem cell, also known as somatic and gametes, refers to any cellwhich is found in a developed organism that has two properties: theability to divide and create another cell like itself and also divideand create a cell more differentiated than itself. They can be found inchildren, as well as adults [Nature (2002), 418(6893), 41-49]. Allsomatic cells of an individual are genetically identical in principle,they evolve a variety of tissue-specific characteristics during theprocess of differentiation, through epigenetic and regulatoryalterations. Pluripotent somatic stem cells are rare and generally smallin number but can be found in a number of tissues including umbilicalcord blood. A great deal of somatic stem cell research has focused onclarifying their capacity to divide or self-renew indefinitely and theirdifferentiation potential. In mice, pluripotent stem cells are directlygenerated from adult fibroblast cultures. Unfortunately, many mice don'tlive long with stem cell organs.

Somatic cells can be reprogrammed to induced pluripotent stem cells(iPSC) by retroviral transduction of four transcription factors [Cell(2008), 132(4), 567-582]. While the reprogrammed pluripotent cells arethought to have great potential for regenerative medicine [Proc. Natl.Acad. Sci. USA (2008), 105(15), 5856-5861], genomic integrations of theretroviruses, especially c-Myc, increase the risk of tumorigenesis[Nature (2007), 448(7151), 313-317]. Generation of iPSCs for use in theclinical setting would benefit from identification of alternative,ultimately safer, initiating stimuli, in preference to geneticmodification. This could be transient treatment with defined factors,low-toxicity chemicals, or synthetic small molecules. Since the Wntpathway is intimately connected to the core circuitry of pluripotency,it has been shown that the stimulation of the pathway using solubleWnt3a promotes the generation of iPSCs in the absence of c-Mycretrovirus. These data demonstrate that signal transduction pathways andtranscription factors can act coordinately to reprogram differentiatedcells to a pluripotent state [Cell Stem Cell (2008), 3(2), 132-135; CellStem Cell (2008), 3(5), 465-466].

As discussed above, activators of the Wnt/β-catenin signaling pathwayare expected to be medicaments useful against cell proliferationdisorders, bone disorders, eye diseases, Alzheimer's disease and eventissue generation. Thus, it would be advantageous to have novelactivators of the Wnt/β-catenin signaling pathway as potential treatmentregimens for Wnt/β-catenin signaling pathway-related disorders. Theinstant invention is directed to these and other important ends.

SUMMARY OF THE INVENTION

The present invention relates to a method for increasing cell or tissueregeneration in a vertebrate subject. The invention relates to methodsfor increasing the successful activity of embryonic and/or adult stemcells, progenitor cells, mesenchymal progenitor/stem cells and/ordifferentiated cells in vivo in a vertebrate subject. The inventionfurther relates to methods for increasing cell or tissue regeneration ina vertebrate subject by administering a compound according to FormulasI, II or III, to the vertebrate subject in need thereof, and increasingin vivo a stem cell, progenitor cell, and/or differentiated cellpopulation in the vertebrate subject compared to the stem cell,progenitor cell, and/or differentiated cell population in the vertebratesubject before treatment, to increase cell or tissue regeneration in thevertebrate subject. Increasing the stem cell, progenitor cell, ordifferentiated cell population in the vertebrate subject can be a resultof cell proliferation, cell homing, decreased apoptosis, self-renewal,or increased cell survival.

In one embodiment, the cell or tissue regeneration can occur in tissuesincluding but not limited to, bone, chondrocytes/cartilage, muscle,skeletal muscle, cardiac muscle, pancreatic cells, endothelial cells,vascular endothelial cells, adipose cells, liver, skin, connectivetissue, hematopoietic stem cells, neonatal cells, umbilical cord bloodcells, fetal liver cells, adult cells, bone marrow cells, peripheralblood cells, erythroid cells, granulocyte cells, macrophage cells,granulocyte-macrophage cells, B cells, T cells, multipotent mixedlineage colony types, embryonic stem cells, mesenchymal progenitor/stemcells, mesodermal progenitor/stem cells, neural progenitor/stem cells,or nerve cells. The vertebrate can be mammalian, avian, reptilian,amphibian, osteichthyes, or chondrichthyes.

In one embodiment, the present invention is a composition for preventingor decreasing the loss of hair and/or for stimulating or increasing hairgrowth or regrowth, wherein the composition comprises a compoundaccording to Formulas I, II or III.

One embodiment of the present invention provides a pharmaceuticalcomposition for the treatment of a neurodegenerative disease.

In another embodiments, the neurological disorder is Alzheimer'sdisease, schizophrenia or schizo-affective disorder, bipolar disorder orunipolar disorder, depression, substance abuse, neurodegenerativedisease, autism or autism spectrum disorder, or a disorder resultingfrom neural damage such as spinal injuries or brain injuries. Theneurodegenerative disease may be for instance, amyotrophic lateralsclerosis (Lou Gehrig's disease) or Parkinson's disease. In someembodiments, the invention provides methods for treating brain injuryresulting from traumatic injury or stroke.

In another embodiments, the neurological disorder is an eye disease suchas age related macular degeneration, diabetic macular edema or retinitispigmentosa.

In one embodiment, the invention relates to a method for (i) reducingloss of bone mass or bone density, (ii) increasing bone mass or bonedensity, (iii) maintaining bone mass or bone density and/or (iv)reducing loss of calcium from bone, comprising: administering to asubject a therapeutically effective amount of a compound according toFormulas I, II or III. As used in this patent specification, the term“bone mass” and “bone density” are used interchangeably.

In one embodiment, the invention relates to a method to regulateosteoblast activity or osteoclast activity comprising the use of acompound according to Formulas I, II or III. Osteoblast activity can beregulated by regulating the proliferation or function of osteoblasts.The function of osteoblasts and/or osteoclasts can be regulated directlyor indirectly.

In one embodiment, the method is for the treatment of a bone conditionor a bone defect.

In another embodiment, the bone condition being treated is frailty, anosteoporotic fracture, a bone defect, childhood idiopathic bone loss,alveolar bone loss, mandibular bone loss, bone fracture, osteotomy, boneloss associated with periodontitis, or prosthetic ingrowth.

In yet another embodiment, the bone condition being treated is Paget'sdisease.

In another embodiment, the bone condition being treated is oncolyticbone disease.

In another embodiment, the invention relates to method for promotinghealing of bone fractures, bone defects, craniofacial defects,otosclerosis or osteogenesis imperfecta comprising: administering to asubject a therapeutically effective amount of a compound according toFormulas I, II or III.

In another embodiment, the invention relates to method for bone tissueengineering comprising the use a compound according to Formulas I, II orIII. In one embodiment the cells used for bone tissue engineering aretreated with a compound according to Formulas I, II or III.

In another embodiment, the invention relates to the use of a compoundaccording to Formulas I, II or III as a medicament for (i) reducing lossof bone mass, (ii) increasing bone mass, (iii) maintaining bone massand/or (iv) reducing loss of calcium from bone in a subject in needthereof. In another embodiment, the invention relates to the use of acompound according to Formulas I, II or III as a medicament for healingbone fractures or repairing bone defects in a mammal.

In one embodiment, the bone condition being treated is osteoporosis. Inone embodiment, the osteoporosis being treated is selected from thegroup consisting of: glucocorticoid-induced osteoporosis,hyperthyroidism-induced osteoporosis, immobilization-inducedosteoporosis, heparin-induced osteoporosis and immunosuppressive-inducedosteoporosis.

In one embodiment, a compound according to Formulas I, II or III isadministered conjointly with an agent that increases bone mass orprevents the loss of bone mass. In one embodiment, the agent thatincreases bone mass is a growth factor, a mineral, a vitamin, a hormone,a prostaglandin, an inhibitor of 15-lipoxygenase, a bone morphogenicprotein or another member of the TGF-beta superfamily which increasesbone formation, an ACE inhibitor, a Hedgehog protein, examethasone,calcitonin, or an active fragment thereof. In one embodiment, the agentthat prevents the loss of bone mass is progestin, estrogen, anestrogen/progestin combinations, estrone, estriol, 17α- or 17β-ethynylestradiol, SB242784, polyphosphonates, biphosphonates or an activefragment thereof.

In one embodiment of the invention, a compound according to Formulas I,II or III, is administered to enhance proliferation of intestinalepithelium, for the treatment, or as a therapeutic adjunct in thetreatment, of diseases that compromise the intestinal epithelia,including inflammatory bowel diseases and Celiac disease.

In another embodiment, the invention relates to a method for organtissue engineering comprising the use of a compound according toFormulas I, II or III. In one embodiment the cells used for organ tissueengineering are treated with a compound according to Formulas I, II orIII.

Some embodiments disclosed herein include a Wnt/β-catenin signalingpathway activator containing a β-diketone, γ-diketone or γ-hydroxyketonecore. Other embodiments disclosed herein include pharmaceuticalcompositions and methods of treatment using these compounds.

One embodiment of a Wnt/β-catenin signaling pathway activator disclosedherein includes a compound having the structure of Formula I:

R¹ is selected from the group consisting of substituted or unsubstitutedheteroaryl and substituted or unsubstituted heterocyclyl, with theproviso that a carbon atom is attached to the carbonyl;

R² is selected from the group consisting of substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted carbocyclyl and substituted or unsubstituted heterocyclyl,with the proviso that a carbon atom is attached to the carbonyl; and

R³, R⁴, R⁵ and R⁶ are independently selected from a group consisting ofH, —C₁₋₉alkyl, —C₁₋₉alkylaryl and —C₁₋₉alkylheteroaryl.

Another embodiment of a Wnt/β-catenin signaling pathway activatordisclosed herein includes a compound having the structure of Formula II:

R¹ is selected from the group consisting of substituted or unsubstitutedheteroaryl and substituted or unsubstituted heterocyclyl, with theproviso that a carbon atom is attached to the carbonyl;

R² is selected from the group consisting of substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted carbocyclyl and substituted or unsubstituted heterocyclyl,with the proviso that a carbon atom is attached to the carbonyl; and

R³ and R⁴ are independently selected from a group consisting of H,—C₁₋₉alkyl, —C₁₋₉alkylaryl and —C₁₋₉alkylheteroaryl.

Another embodiment of a Wnt/β-catenin signaling pathway activatordisclosed herein includes a compound having the structure of FormulaIII:

R¹ is selected from the group consisting of substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted carbocyclyl and substituted or unsubstituted heterocyclyl,with the proviso that a carbon atom is attached to the carbonyl;

R² is selected from the group consisting of substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted carbocyclyl and substituted or unsubstituted heterocyclyl,with the proviso that a carbon atom is attached to the carbonyl; and

R³ and R⁴ are independently selected from a group consisting of H,—C₁₋₉alkyl, —C₁₋₉alkylaryl and —C₁₋₉alkylheteroaryl.

Some embodiments include stereoisomers and pharmaceutically acceptablesalts of a compound of Formulas I, II or III.

Some embodiments include pro-drugs of a compound of Formulas I, II andIII.

Some embodiments of the present invention include pharmaceuticalcompositions comprising a compound of Formulas I, II or III and apharmaceutically acceptable carrier.

Another embodiment disclosed herein includes a pharmaceuticalcomposition comprising a compound according to any of the above formulasand a pharmaceutically acceptable carrier, diluent, or excipient.

Some embodiments of the present invention include methods to preparecompounds of Formulas I, II or III.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

DETAILED DESCRIPTION OF THE INVENTION

It has been discovered that β-diketones, γ-diketones andγ-hydroxyketones are capable of activating the Wnt/β-catenin signalingpathway. The Wnt/β-catenin signaling pathway has been found to play acrucial role in the differentiation and development of nerve cells forthe central nervous system, bone formation, hair follicle developmentand regeneration, and stimulation of stem cell growth, maintenance anddifferentiation.

The present invention relates a method for increasing cell or tissueregeneration in a vertebrate subject. The invention relates to methodsfor increasing the successful activity of embryonic and/or adult stemcells, progenitor cells, mesenchymal progenitor/stem cells, ordifferentiated cells in vivo in a vertebrate subject. The inventionfurther relates to methods for increasing cell or tissue regeneration ina vertebrate subject by administering a compound according to FormulasI, II or III to the vertebrate subject in need thereof, and increasingin vivo a stem cell, progenitor cell population, or differentiated cellin the vertebrate subject compared to the stem cell or progenitor cell,or differentiated cell population in the vertebrate subject beforetreatment, to increase cell or tissue regeneration in the vertebratesubject. A method for increasing stem cell or progenitor cell populationis provided to repair or replace damaged tissue in a vertebrate subject,wherein the cell or tissue regeneration occurs in bone,chondrocytes/cartilage, muscle, skeletal muscle, cardiac muscle,pancreatic cells, endothelial cells, vascular endothelial cells, adiposecells, liver, skin, connective tissue, hematopoietic stem cells,neonatal cells, umbilical cord blood cells, fetal liver cells, adultcells, bone marrow cells, peripheral blood cells, erythroid cells,granulocyte cells, macrophage cells, granulocyte-macrophage cells, Bcells, T cells, multipotent mixed lineage colony types, embryonic stemcells, mesenchymal progenitor/stem cells, mesodermal progenitor/stemcells, neural progenitor/stem cells, or nerve cells.

Hair Growth

Compositions comprising compounds according to Formulas I, II or III canbe used to promote hair growth.

“Promoting hair growth” refers to maintaining, inducing, stimulating,accelerating, or revitalizing the germination of hair.

The method of the present invention is useful in the treatment ofalopecia in mammals, and as such may be used to promote, increase, orassist in the growth of hair. Subjects may be male or female. The termalopecia refers to both the complete absence of hair in skin whichtypically exhibits hair growth, as well as to a loss or diminution inthe amount of hair. Multiple types and causes of alopecia are recognizedin humans, including male pattern baldness, chemotherapy induced hairloss, congenital alopecia, and alopecia areata. The term treatingalopecia refers to both the treatment of skin with a total absence ofhair growth as well as the treatment of skin having reduced or patchyhair growth. Successful treatment results in an increased number ofhairs.

Subjects to be treated according to the invention include human subjectsas well as other mammalian subjects, such as dogs, cats, mice, rats,goats, llamas, minks, seals, beavers, ermines, and sheep. These can betreated for hair loss due or simply for enhancing wool or peltproduction.

“Treating alopecia” refers to (i) preventing alopecia in an animal whichmay be predisposed to alopecia, (ii) inhibiting, retarding or reducingalopecia, (iii) promoting hair growth and/or (iv) prolonging the anagenphase of the hair cycle.

A method for promoting hair growth in accordance with the presentinvention is characterized by applying an effective amount of a compoundaccording to Formulas I, II or III, or a pharmacologically acceptablesalt thereof on the skin of mammals and in particular, on human scalp.

Neurological Disorder

Compounds according to the present invention can modulate the cellularfate of neural stem cells and promote the differentiation of theseneural precursors to functional neurons and glial cells.

Compositions comprising compounds according to Formulas I, II or III canbe used to treat neurodegenerative diseases.

Non-limiting examples of neurodegenerative diseases are Alzheimer'sdisease, schizophrenia or schizo-affective disorder, bipolar disorder orunipolar disorder, depression, substance abuse, neurodegenerativedisease, autism or autism spectrum disorder, or a disorder resultingfrom neural damage such as spinal injuries or brain injuries. Theneurodegenerative disease may be for instance, amyotrophic lateralsclerosis (Lou Gehrig's disease) or Parkinson's disease.

Other non-limiting examples of neurodegenerative diseases are eyediseases such as age related macular degeneration, diabetic macularedema or retinitis pigmentosa.

The invention also provides a method for treating brain injury resultingfrom traumatic injury or stroke.

Another aspect of the invention is a method of enhancing neuralprogenitor proliferation and differentiation by contacting a neuralprogenitor cell with a compound according to Formulas I, II or III in aneffective amount to enhance neural progenitor proliferation anddifferentiation.

In one aspect the invention provides a method of enhancing nervegeneration, by contacting a nerve with a compound according to FormulasI, II or III in an effective amount to enhance nerve generation.

In another aspect, the present invention provides a method of treating aneurodegenerative disease in a patient requiring treatment, whichcomprises administering an effective amount of a compound of Formulas I,II or III, or a pharmaceutically acceptable salt thereof as definedhereinabove.

The compounds according to the present invention may be administeredalone or co-administered with compounds working by a differentmechanism, for example neuroprotectant agents. In one embodiment, thecompounds are co-administered compounds with an acetylcholinesteraseinhibitor (e.g. Aricept) for Alzheimer's disease or L-DOPA for Parkinsondisease.

Bone Formation

Compositions comprising compounds of Formulas I, II or III can be usedto treat, prevent and alleviate bone conditions. The present inventionprovides a method for (i) reducing loss of bone mass, (ii) increasingbone mass, (iii) maintaining bone mass and/or (iv) reducing loss ofcalcium from bone, comprising: administering to a subject atherapeutically effective amount of a compound according to Formulas I,II or III. The method could be used for treating, preventing or delayinga bone condition. The invention further provides a method for promotinghealing of bone fractures or bone defects comprising: administering to asubject a therapeutically effective amount of a compound according toFormulas I, II or III. Any of the above mentioned methods can involvethe conjoint administration of an agent that increases bone mass orprevents the loss of bone mass.

The invention also provides for the use of a compound according toFormulas I, II or III as a medicament for treating, preventing ordelaying a bone condition.

As used herein, the term “bone condition” includes any condition whereit is desirable to increase bone mass or bone density and/or prevent theloss of bone mass or bone density. A bone condition includes anycondition that increases osteoclast number, increases osteoclastactivity, increases bone resorption, increases marrow fibrosis, oralters the calcium content of bone.

Non-limiting examples of bone conditions include metabolic boneconditions such as renal osteodystrophy, primary forms of osteoporosis(e.g., postmenopausal and senile osteoporosis), and secondary forms ofosteoporosis that develop as a result of an underlying disease state.For example, osteoporosis can develop in patients that have endocrinedisorders such as hyperparathyroidism, hypo- and hyperthyroidism,hypogonadism, hypercalcemia due to malignancy, pituitary tumors, type Idiabetes, or Addison's disease. Neoplasias such as multiple myeloma andcarcinomatosis also can lead to development of osteoporosis. Inaddition, gastrointestinal problems such as malnutrition, malabsorption,hepatic insufficiency, and vitamin C or D deficiencies, and chronicadministration of drugs such as anticoagulants, chemotherapeutics,corticosteroids, anticonvulsants, and alcohol can lead to development ofosteoporosis.

Non-limiting examples of bone conditions also include osteonecrosis,osteoarthritis, rheumatoid arthritis, Paget's disease, osteogenesisimperfecta, chronic hyperparathyroidism, hyperthyroidism, Gorham-Stoutdisease, McCune-Albright syndrome, and alveolar ridge bone loss.

The term “bone condition” includes, without limitation, all conditionsresulting in bone loss, including, cancers and tumors (such asosteosarcoma and multiple myeloma), renal disease (including acute renalfailure, chronic renal failure, renal bone dystrophy and renalreperfusion injury), kidney disease, premature ovarian failure and otherconditions.

Endocrine disorders, vitamin deficiencies and viral infections also canlead to development of bone conditions that can be treated with methodsof the invention. An example of a bone condition caused by a nutritionaldisorder is osteomalacia, a nutritional disorder caused by a deficiencyof vitamin D and calcium. It is referred to as “rickets” in children,and “osteomalacia” in adults. It is marked by a softening of the bones(due to impaired mineralization, with excess accumulation of osteoid),pain, tenderness, muscle wasting and weakness, anorexia, and overallweight loss. It can result from malnutrition, repeated pregnancies andlactation (exhausting or depleting vitamin D and calcium stores), andvitamin D resistance.

Bone conditions include conditions resulting from the treatment of asubject with drugs, for example the osteopenia resulting from thetreatment with Cyclosporin A or FK506.

Bone conditions also include bone fractures, bone trauma, conditionsassociated with post-traumatic bone surgery, post-prosthetic jointsurgery, post-plastic bone surgery, post-dental surgery, bonechemotherapy, post-dental surgery and bone radiotherapy. Fracturesinclude all types of microscopic and macroscopic fractures. Examples offractures includes avulsion fracture, comminuted fracture, transversefracture, oblique fracture, spiral fracture, segmental fracture,displaced fracture, impacted fracture, greenstick fracture, torusfracture, fatigue fracture, intra-articular fracture (epiphysealfracture), closed fracture (simple fracture), open fracture (compoundfracture) and occult fracture.

Other non-limiting examples of bone conditions include bone deformation,spinal deformation, prosthesis loosening, bone dysplasia, scoliosis,periodontal disease and defects, tooth repair, and fibrous osteitis.

The invention also provides a method for treating a subject with atherapeutically effective amount of a compound according to Formulas I,II or III, wherein the subject is in need of bone repair followingsurgery, such as cranio-maxillofacial repair following tumor removal,surgical bone reconstruction following traumatic injury, repair ofhereditary or other physical abnormalities, and promotion of bonehealing in plastic surgery.

The invention also provides a method for treating a subject with atherapeutically effective amount of a compound according to Formulas I,II or III, wherein the subject is in need of bone repair after receivingan implant (including joint replacements and dental implants), aprosthesis or a bone graft.

The invention also provides a method for treating a subject with atherapeutically effective amount of a compound according to Formulas I,II or III, wherein the subject: a) is in need of increased bone densityor bone healing; b) has undergone or is presently undergoingcorticosteroid therapy, dialysis, chemotherapy for post-menopausal boneloss, radiation therapy for cancer or hormone replacement therapy; c) isimmobilized or subjected to extended bed rest due to bone injury; d)suffers from alcoholism, diabetes, hyperprolactinemia, anorexia nervosa,primary and secondary amenorrhea, or oophorectomy; e) suffers from renalfailure; f) is 50 years or older; or g) is a female.

The invention also provides a method for treating a subject with atherapeutically effective amount of a compound according to Formulas I,II or III, wherein the subject is affected by a disease selected fromarterial calcification, ankylosing spondylitis, ossification of theposterior longitudinal ligament, myositis ossificans, diffuse idiopathicskeletal hyperostosis, calcific tendonitis, rotator cuff disease of theshoulders, bone spurs, cartilage or ligament degeneration due tohydroxyapatite crystal deposition, and chondrocalcinosis.

By the term “effective amount” or “therapeutically effective amount” ofa compound according to Formulas I, II or III, is meant an amountsufficient to obtain the desired physiological effect, e.g., activationof osteoblasts, increase in osteoblast number, increase in boneformation, a decrease in osteoclasts number or the deactivation ofosteoclasts. An effective amount of a Wnt/β-catenin signaling pathwayactivator is determined by the care giver in each case on the basis offactors normally considered by one skilled in the art to determineappropriate dosages, including the age, sex, and weight of the subjectto be treated, the condition being treated, and the severity of themedical condition being treated.

The invention also provides a method for treating a subject with atherapeutically effective amount of a compound according to Formulas I,II or III conjointly with an agent that increases bone mass or preventsthe loss of bone mass. In one embodiment, the agent that increases bonemass is a growth factor, a mineral, a vitamin, a hormone, aprostaglandin, an inhibitor of 15-lipoxygenase, a bone morphogenicprotein or another member of the TGF-beta superfamily which increasesbone formation, an ACE inhibitor, a Hedgehog protein, examethasone,calcitonin, or an active fragment thereof. In one embodiment, the agentthat prevents the loss of bone mass is progestin, estrogen, anestrogen/progestin combinations, estrone, estriol, 17α- or 17β-ethynylestradiol, SB242784, polyphosphonates, biphosphonates or an activefragment thereof.

Intestinal Diseases

Compounds according to Formulas I, II or III are also administered forthe treatment of gastrointestinal inflammation. “Gastrointestinalinflammation” as used herein refers to inflammation of a mucosal layerof the gastrointestinal tract, and encompasses acute and chronicinflammatory conditions. Acute inflammation is generally characterizedby a short time of onset and infiltration or influx of neutrophils.

“Chronic gastrointestinal inflammation” refers to inflammation of themucosal of the gastrointestinal tract that is characterized by arelatively longer period of onset, is long-lasting (e. g., from severaldays, weeks, months, or years and up to the life of the subject), and isassociated with infiltration or influx of mononuclear cells and can befurther associated with periods of spontaneous remission and spontaneousoccurrence. Thus, subjects with chronic gastrointestinal inflammationmay be expected to require a long period of supervision, observation, orcare. “Chronic gastrointestinal inflammatory conditions” (also referredto as “chronic gastrointestinal inflammatory diseases”) having suchchronic inflammation include, but are not necessarily limited to,inflammatory bowel disease (IBD), colitis induced by environmentalinsults (e. g., gastrointestinal inflammation (e. g., colitis) caused byor associated with (e. g., as a side effect) a therapeutic regimen, suchas administration of chemotherapy, radiation therapy, and the like),colitis in conditions such as chronic granulomatous disease, celiacdisease, celiac sprue (a heritable disease in which the intestinallining is inflamed in response to the ingestion of a protein known asgluten), food allergies, gastritis, infectious gastritis orenterocolitis (e. g., Helicobacter pylori-infected chronic activegastritis) and other forms of gastrointestinal inflammation caused by aninfectious agent, and other like conditions.

As used herein, “inflammatory bowel disease” or “IBD” refers to any of avariety of diseases characterized by inflammation of all or part of theintestines. Examples of inflammatory bowel disease include, but are notlimited to, Crohn's disease and ulcerative colitis. Reference to IBDthroughout the specification is often referred to in the specificationas exemplary of gastrointestinal inflammatory conditions, and is notmeant to be limiting.

Compounds according to Formulas I, II or III can be administered to asubject prior to onset of more severe symptoms (e. g., prior to onset ofan acute inflammatory attack), or after onset of acute or chronicsymptoms (e. g., after onset of an acute inflammatory attack). As such,the agents can be administered at any time, and may be administered atany interval. In one embodiment, compounds according to Formulas I, IIor III are administered about 8 hours, about 12 hours, about 24 hours,about 2 days, about 4 days, about 8 days, about 16 days, about 30 daysor 1 month, about 2 months, about 4 months, about 8 months, or about 1year after initial onset of gastrointestinal inflammation-associatedsymptoms and/or after diagnosis of gastrointestinal inflammation in thesubject.

When multiple doses are administered, subsequent doses are administeredwithin about 16 weeks, about 12 weeks, about 8 weeks, about 6 weeks,about 4 weeks, about 2 weeks, about 1 week, about 5 days, about 72hours, about 48 hours, about 24 hours, about 12 hours, about 8 hours,about 4 hours, or about 2 hours or less of the previous dose. In oneembodiment, ISS are administered at intervals ranging from at leastevery two weeks to every four weeks (e. g., monthly intervals) in orderto maintain the maximal desired therapeutic effect (e. g., to providefor maintenance of relief from BD-associated symptoms).

Regenerative Medicine

According to the present invention, somatic cells can be provided thatare capable as serving as a primitive organ-like structure comprised ofa plurality of types of somatic cell types.

Somatic cells as referred to in the present invention refer to cellsthat have reached differentiation into cells that compose various organsof the body, and refer to cells that are the opposite ofundifferentiated stem cells. The present invention is characterized bythe use of two or more somatic cells, and preferably consists of variouscombinations thereof, such as a combination of an epithelial cell lineand mesenchymal cells, a combination of endothelial cells andmesenchymal cells, or a combination of epithelial cells and mesenchymalcells.

There are no particular limitations on organs capable of being formed bythe somatic cells as claimed in the present invention, examples of whichinclude various organs such as hair follicle, lung, kidney, liver,pancreas, spleen, heart, gallbladder, small intestine, colon, largeintestine, joint, bone, tooth, blood vessel, lymph duct, cornea,cartilage, olfactory organ or auditory organ.

Various mammals can be used without limitation as the origin of thecells as claimed in the present invention corresponding to the purposethereof, examples of which include chimpanzees, other primates, domesticanimals such as dogs or cats, farm animals such as cows, pigs, horses,sheep or goats, laboratory animals such as rabbits, rats, mice or guineapigs, and more preferably nude mice, SCID mice or nude rats. Inaddition, although combinations thereof may be homogeneous combinationsor heterogeneous combinations, homogeneous combinations are preferable.

The present invention is characterized by the addition of aWnt/β-catenin signaling pathway activator according to Formulas I, II orIII to a mixture of types of differentiated somatic cells as describedabove followed by culturing thereof. Wnt signaling refers to a series ofactions that demonstrate the function of transcription factors bypromoting nuclear migration of β-catenin. These signals originate fromcellular interaction that includes, for example, a series of processesin which a protein referred to as Wnt3A secreted from certain cellsfurther acts on other cells causing nuclear migration of intracellularβ-catenin which acts as a transcription factor. This series of processesgive rise to the initial phenomenon of organ construction in the exampleof epithelial-mesenchymal interaction. The Wnt/β-catenin signalingpathway is known to control cell proliferation and differentiation,organ formation and various cell functions such as cell migration duringinitial development. Although Wnt signaling is used when culturing EScells for the purpose of inhibiting differentiation due to theirfunction of maintaining an undifferentiated state, their utilization andeffects during culturing of somatic cells are completely unknown.

Another characteristic of the present invention is the subjecting of themixture of types of differentiated somatic cells, to which a compoundaccording to Formulas I, II or III has been added, to non-plate contactculturing. Non-plate contact culturing refers to a method of culturingcells on an interface having a spherical surface so as not to allowadhesion of plate-adhering cells. An example of non-plate contactculturing is a hanging drop method. The hanging drop method refers toadhering a drop of culture medium containing cultured cells onto theinside of the upper lid of a culture dish, carefully closing the lid sothat the culture medium does not drop or run down, and culturing cellswithin the culture medium to be cultured in the form of an inverted dropdue to surface tension. As a result of culturing in this manner, theeffects on the cells attributable to contact with a flat surface as inthe case of plate culturing can be minimized. Other examples ofnon-plate contact culturing methods include a formation method utilizinga semi-spherical cell culture dish that has been surface-treated inadvance to prevent cell adhesion (for example, “Spheroid” commerciallyavailable from Sumitomo Bakelite) (referred to as the spheroid formationmethod), and a suspension method in which cells are aggregated in asuspended state by culturing in a nitrocellulose medium.

In some embodiments, pharmaceutical compositions are provided that areeffective for treatment of a disease of an animal, e.g., a mammal,caused by the pathological activation or mutations of the Wnt pathway.The composition includes a pharmaceutically acceptable carrier and a Wntpathway activator as described herein.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this disclosure belongs. All patents, applications,published applications, and other publications are incorporated byreference in their entirety. In the event that there is a plurality ofdefinitions for a term herein, those in this section prevail unlessstated otherwise.

In this specification and in the claims, the following terms have themeanings as defined. As used herein, “alkyl” means a branched, orstraight chain chemical group containing only carbon and hydrogen, suchas methyl, isopropyl, isobutyl, sec-butyl and pentyl. Alkyl groups caneither be unsubstituted or substituted with one or more substituents,e.g., halogen, alkoxy, acyloxy, amino, amido, cyano, nitro, hydroxyl,mercapto, carboxy, carbonyl, benzyloxy, aryl, heteroaryl, or otherfunctionality that may be suitably blocked, if necessary for purposes ofthe invention, with a protecting group. Alkyl groups can be saturated orunsaturated (e.g., containing —C═C— or —C≡C— subunits), at one orseveral positions. Typically, alkyl groups will comprise 1 to 9 carbonatoms, preferably 1 to 6, and more preferably 1 to 4 carbon atoms.

As used herein, “carbocyclyl” means a cyclic ring system containing onlycarbon atoms in the ring system backbone, such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, and cyclohexenyl. Carbocyclyls mayinclude multiple fused rings. Carbocyclyls may have any degree ofsaturation provided that at least one ring in the ring system is notaromatic. Carbocyclyl groups can either be unsubstituted or substitutedwith one or more substituents, e.g., halogen, alkoxy, acyloxy, amino,amido, cyano, nitro, hydroxyl, mercapto, carboxy, carbonyl, benzyloxy,aryl, heteroaryl, or other functionality that may be suitably blocked,if necessary for purposes of the invention, with a protecting group.Typically, carbocyclyl groups will comprise 3 to 10 carbon atoms,preferably 3 to 6.

As used herein, “lower alkyl” means a subset of alkyl, and thus is ahydrocarbon substituent, which is linear, or branched. Preferred loweralkyls are of 1 to about 4 carbons, and may be branched or linear.Examples of lower alkyl include butyl, propyl, isopropyl, ethyl, andmethyl. Likewise, radicals using the terminology “lower” refer toradicals preferably with 1 to about 4 carbons in the alkyl portion ofthe radical.

As used herein, “amido” means a H—CON— or alkyl-CON—, carbocyclyl-CON—,aryl-CON—, heteroaryl-CON— or heterocyclyl-CON group wherein the alkyl,carbocyclyl, aryl or heterocyclyl group is as herein described.

As used herein, “aryl” means an aromatic radical having a single-ring(e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl)with only carbon atoms present in the ring backbone. Aryl groups caneither be unsubstituted or substituted with one or more substituents,e.g., amino, cyano, hydroxyl, lower alkyl, haloalkyl, alkoxy, nitro,halo, mercapto, and other substituents. A preferred carbocyclic aryl isphenyl.

As used herein, the term “heteroaryl” means an aromatic radical havingone or more heteroatom(s) (e.g., N, O, or S) in the ring backbone andmay include a single ring (e.g., pyridine) or multiple condensed rings(e.g., quinoline). Heteroaryl groups can either be unsubstituted orsubstituted with one or more substituents, e.g., amino, cyano, hydroxyl,lower alkyl, haloalkyl, alkoxy, nitro, halo, mercapto, and othersubstituents. Examples of heteroaryl include thienyl, pyrridyl, furyl,oxazolyl, oxadiazolyl, pyrollyl, imidazolyl, triazolyl, thiodiazolyl,pyrazolyl, isoxazolyl, thiadiazolyl, pyranyl, pyrazinyl, pyrimidinyl,pyridazinyl, triazinyl, thiazolyl and others.

In these definitions it is clearly contemplated that substitution on thearyl and heteroaryl rings is within the scope of certain embodiments.Where substitution occurs, the radical is called substituted aryl orsubstituted heteroaryl. Preferably one to three and more preferably oneor two substituents occur on the aryl ring. Though many substituentswill be useful, preferred substituents include those commonly found inaryl compounds, such as alkyl, cycloalkyl, hydroxy, alkoxy, cyano, halo,haloalkyl, mercapto and the like.

As used herein, “amide” includes both RNR′CO— (in the case of R=alkyl,alkaminocarbonyl-) and RCONR′— (in the case of R=alkyl, alkylcarbonylamino-).

As used herein, the term “ester” includes both ROCO— (in the case ofR=alkyl, alkoxycarbonyl-) and RCOO— (in the case of R=alkyl,alkylcarbonyloxy-).

As used herein, “acyl” means an H—CO— or alkyl-CO—,carbocyclyl-CO-aryl-CO—, heteroaryl-CO— or heterocyclyl-CO— groupwherein the alkyl, carbocyclyl, aryl or heterocyclyl group is as hereindescribed. Preferred acyls contain a lower alkyl. Exemplary alkyl acylgroups include formyl, acetyl, propanoyl, 2-methylpropanoyl,t-butylacetyl, butanoyl and palmitoyl.

As used herein, “halo or halide” is a chloro, bromo, fluoro or iodo atomradical. Chloro, bromo and fluoro are preferred halides. The term “halo”also contemplates terms sometimes referred to as “halogen”, or “halide”.

As used herein, “haloalkyl” means a hydrocarbon substituent, which islinear or branched or cyclic alkyl, alkenyl or alkynyl substituted withchloro, bromo, fluoro or iodo atom(s). Most preferred of these arefluoroalkyls, wherein one or more of the hydrogen atoms have beensubstituted by fluoro. Preferred haloalkyls are of 1 to about 3 carbonsin length, more preferred haloalkyls are 1 to about 2 carbons, and mostpreferred are 1 carbon in length. The skilled artisan will recognizethen that as used herein, “haloalkylene” means a diradical variant ofhaloalkyl, such diradicals may act as spacers between radicals, otheratoms, or between the parent ring and another functional group.

As used herein, “heterocyclyl” means a cyclic ring system comprising atleast one heteroatom in the ring system backbone. Heterocyclyls mayinclude multiple fused rings. Heterocyclyls may have any degree ofsaturation provided that at least one ring in the ring system is notaromatic. Heterocyclyls may be substituted or unsubstituted with one ormore substituents, e.g., halogen, alkoxy, acyloxy, amino, amido, cyano,nitro, hydroxyl, mercapto, carboxy, carbonyl, benzyloxy, aryl,heteroaryl, and other substituents, and are attached to other groups viaany available valence, preferably any available carbon or nitrogen. Morepreferred heterocycles are of 5-7 members. In six membered monocyclicheterocycles, the heteroatom(s) are selected from one up to three of O,N or S, and wherein when the heterocycle is five membered, preferably ithas one or two heteroatoms selected from O, N, or S.

As used herein, “substituted amino” means an amino radical which issubstituted by one or two alkyl, cycloalkyl, aryl, heteroaryl orheterocyclyl groups, wherein the alkyl, aryl, heteroaryl or heterocyclylare defined as above.

As used herein, “substituted thiol” means RS— group wherein R is analkyl, an aryl, heteroaryl or a heterocyclyl group, wherein the alkyl,cycloalkyl, aryl, heteroaryl or heterocyclyl are defined as above.

As used herein, “sulfonyl” means an alkylSO₂, arylSO₂, heteroarylSO₂,carbocyclylSO₂, or heterocyclyl-SO₂ group wherein the alkyl,carbocyclyl, aryl, heteroaryl or heterocyclyl are defined as above.

As used herein, “sulfamido” means an alkyl-N—S(O)₂N—,aryl-NS(O)₂N-heteroaryl-NS(O)₂N—, carbocyclyl-NS(O)₂N orheterocyclyl-NS(O)₂N— group wherein the alkyl, carbocyclyl, aryl,heteroaryl or heterocyclyl group is as herein described.

As used herein, “sulfonamido” means an alkyl-S(O)₂N—, aryl-S(O)₂N—,heteroaryl-S(O)₂N—, carbocyclyl-S(O)₂N— or heterocyclyl-S(O)₂N— groupwherein the alkyl, carbocyclyl, aryl, heteroaryl or heterocyclyl groupis as herein described.

As used herein, “ureido” means an alkyl-NCON—, aryl-NCON—,heteroaryl-NCON—, carbocyclyl-NCON— or heterocyclyl-NCON— group whereinthe alkyl, carbocyclyl, aryl, heteroaryl or heterocyclyl group is asherein described.

As used herein, when two groups are indicated to be “linked” or “bonded”to form a “ring,” it is to be understood that a bond is formed betweenthe two groups and may involve replacement of a hydrogen atom on one orboth groups with the bond, thereby forming a carbocyclyl, heterocyclyl,aryl, or heteroaryl ring. The skilled artisan will recognize that suchrings can and are readily formed by routine chemical reactions, and itis within the purview of the skilled artisan to both envision such ringsand the methods of their formations. Preferred are rings having from 3-7members, more preferably 5 or 6 members. As used herein the term “ring”or “rings” when formed by the combination of two radicals refers toheterocyclic, carbocyclic, aryl, or heteroaryl rings.

The skilled artisan will recognize that some structures described hereinmay be resonance forms or tautomers of compounds that may be fairlyrepresented by other chemical structures, even when kinetically, theartisan recognizes that such structures are only a very small portion ofa sample of such compound(s). Such compounds are clearly contemplatedwithin the scope of this invention, though such resonance forms ortautomers are not represented herein.

The compounds provided herein may encompass various stereochemicalforms. The compounds also encompasses diastereomers as well as opticalisomers, e.g. mixtures of enantiomers including racemic mixtures, aswell as individual enantiomers and diastereomers, which arise as aconsequence of structural asymmetry in certain compounds. Separation ofthe individual isomers or selective synthesis of the individual isomersis accomplished by application of various methods which are well knownto practitioners in the art. Unless otherwise indicated, when adisclosed compound is named or depicted by a structure withoutspecifying the stereochemistry and has one or more chiral centers, it isunderstood to represent all possible stereoisomers of the compound.

The term “administration” or “administering” refers to a method ofgiving a dosage of a compound or pharmaceutical composition to avertebrate or invertebrate, including a mammal, a bird, a fish, or anamphibian, where the method is, e.g., intrarespiratory, topical, oral,intravenous, intraperitoneal, intramuscular, buccal, rectal, sublingual.The preferred method of administration can vary depending on variousfactors, e.g., the components of the pharmaceutical composition, thesite of the disease, the disease involved, and the severity of thedisease.

A “diagnostic” as used herein is a compound, method, system, or devicethat assists in the identification and characterization of a health ordisease state. The diagnostic can be used in standard assays as is knownin the art.

The term “mammal” is used in its usual biological sense. Thus, itspecifically includes humans, cattle, horses, monkeys, dogs, and cats,but also includes many other species.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions. In addition, various adjuvants such as arecommonly used in the art may be included. These and other such compoundsare described in the literature, e.g., in the Merck Index, Merck &Company, Rahway, N.J. Considerations for the inclusion of variouscomponents in pharmaceutical compositions are described, e.g., in Gilmanet al. (Eds.) (2006); Goodman and Gilman's: The Pharmacological Basis ofTherapeutics, 11th Ed., The McGraw-Hill Companies.

The term “pharmaceutically acceptable salt” refers to salts that retainthe biological effectiveness and properties of the compounds of thepreferred embodiments and, which are not biologically or otherwiseundesirable. In many cases, the compounds of the preferred embodimentsare capable of forming acid and/or base salts by virtue of the presenceof amino and/or carboxyl groups or groups similar thereto.Pharmaceutically acceptable acid addition salts can be formed withinorganic acids and organic acids. Inorganic acids from which salts canbe derived include, for example, hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acidsfrom which salts can be derived include, for example, acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and thelike. Pharmaceutically acceptable base addition salts can be formed withinorganic and organic bases. Inorganic bases from which salts can bederived include, for example, sodium, potassium, lithium, ammonium,calcium, magnesium, iron, zinc, copper, manganese, aluminum, and thelike; particularly preferred are the ammonium, potassium, sodium,calcium and magnesium salts. Organic bases from which salts can bederived include, for example, primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines, basic ion exchange resins, and the like, specificallysuch as isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, and ethanolamine. Many such salts are known in the art,as described in World Patent Publication 87/05297, Johnston et al.,published Sep. 11, 1987 (incorporated by reference herein).

“Solvate” refers to the compound formed by the interaction of a solventand a Wnt pathway inhibitor, a metabolite, or salt thereof. Suitablesolvates are pharmaceutically acceptable solvates including hydrates.

“Subject” as used herein, means a human or a non-human mammal, e.g., adog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-humanprimate or a bird, e.g., a chicken, as well as any other vertebrate orinvertebrate.

By “therapeutically effective amount” or “pharmaceutically effectiveamount” is typically one which is sufficient to achieve the desiredeffect and may vary according to the nature and severity of the diseasecondition, and the potency of the compound. It will be appreciated thatdifferent concentrations may be employed for prophylaxis than fortreatment of an active disease. This amount can further depend upon thepatient's height, weight, sex, age and medical history.

A therapeutic effect relieves, to some extent, one or more of thesymptoms of the disease, and includes curing a disease. “Curing” meansthat the symptoms of active disease are eliminated. However, certainlong-term or permanent effects of the disease may exist even after acure is obtained (such as extensive tissue damage).

“Treat,” “treatment,” or “treating,” as used herein refers toadministering a pharmaceutical composition for therapeutic purposes. Theterm “therapeutic treatment” refers to administering treatment to apatient already suffering from a disease thus causing a therapeuticallybeneficial effect, such as ameliorating existing symptoms, preventingadditional symptoms, ameliorating or preventing the underlying metaboliccauses of symptoms, postponing or preventing the further development ofa disorder and/or reducing the severity of symptoms that will or areexpected to develop.

The expression “drug-eluting” shall be understood to refer to any andall mechanisms, e.g., diffusion, migration, permeation, and/ordesorption by which the drug(s) incorporated in the drug-elutingmaterial pass therefrom over time into the surrounding body tissue.

The expression “drug-eluting material” shall be understood herein tomean any natural, synthetic or semi-synthetic material capable ofacquiring and retaining a desired shape or configuration and into whichone or more drugs can be incorporated and from which incorporateddrug(s) are capable of eluting over time.

The expression “elutable drug” shall be understood to mean any drug orcombination of drugs having the ability to pass over time from thedrug-eluting material in which it is incorporated into the surroundingareas of the body.

The following abbreviations have the indicated meanings:

Aβ=amyloid beta

ACE=angiotensin I-converting enzyme

AD=Alzheimer's disease

ALS=amyotrophic Lateral Sclerosis

AMD=age related macular degeneration

APC=adenomatous polyposis coli

β-TrCP=β-transducin repeat-containing protein

CD44=cell-surface glycoprotein

CK1,2=casein kinase 1 and 2

DHT=dihydrotestosterone

Dkk=dickkopf

DME=diabetic macular edema

Dsh/Dvl=dishevelled

EphB2=ephrin type-B receptor 2

ES cells=embryonic stem cells

FTD=frontotemporal Dementia

Fzd=frizzled

GBP=GSK-3 binding protein

GI=gastrointestinal

GPCR=G protein-coupled receptor

GSK-3=glycogen synthase kinase-3

HCC=hepatocellular carcinoma

IBD=inflammatory bowel disease

Kr2=kringle domain 2

L-DOPA=L-3,4-dihydroxyphenylalanine

Lef=lymphoid enhancing factor

LRP=low density lipoprotein receptor related protein

MMTV=mouse mammary tumor virus

PD=Parkinson's disease

PKC=protein kinase C

PI-3=phosphatidylinositol-3 kinase

PPAR=peroxisome proliferator-activated receptors

PTEN=phosphatase and tensin homolog

RP=retinitis pigmentosa

SCID=severe combined immunodeficiency

SOD1=Superoxide dismutase protein

SOST=sclerostin

sFRP=secreted frizzled-related protein

TCF=T-cell factor

TGF=transforming growth factor

UC=ulcerative colitis

Wg=wingless

Wnt=wingless-type MMTV integration site family member

Compounds

The compounds and compositions described herein are capable ofactivating the Wnt/β-catenin signaling pathway. The Wnt/β-cateninsignaling pathway has been found to play a crucial role in thedifferentiation and development of nerve cells for the central nervoussystem, bone formation, hair follicle development and regeneration, andstimulation of stem cell growth, maintenance and differentiation. Suchcompounds and compositions are therefore expected to be useful againstcell proliferation disorders, bone disorders, Alzheimer's disease andeven tissue generation.

Some embodiments of the present invention include compounds, salts,pharmaceutically acceptable salts or pro-drug thereof of formula (I):

In some embodiments, R¹ is selected from the group consisting ofsubstituted or unsubstituted heteroaryl and substituted or unsubstitutedheterocyclyl, with the proviso that a carbon atom is attached to thecarbonyl.

In some embodiments, R² is selected from the group consisting ofsubstituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted carbocyclyl and substituted orunsubstituted heterocyclyl, with the proviso that a carbon atom isattached to the carbonyl.

In some embodiments, R³, R⁴, R⁵ and R⁶ are independently selected from agroup consisting of H, —C₁₋₉alkyl, —C₁₋₉alkylaryl and—C₁₋₉alkylheteroaryl.

In more specific embodiments, R¹ is selected from the group consistingof

In another specific embodiments, R² is selected from the groupconsisting of substituted or unsubstituted aryl,

In another specific embodiments, R³, R⁴, R⁵ and R⁶ are H.

Some embodiments of the present invention include compounds, salts,pharmaceutically acceptable salts or pro-drug thereof of formula (II):

In some embodiments, R¹ is selected from the group consisting ofsubstituted or unsubstituted heteroaryl and substituted or unsubstitutedheterocyclyl, with the proviso that a carbon atom is attached to thecarbonyl.

In some embodiments, R² is selected from the group consisting ofsubstituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted carbocyclyl and substituted orunsubstituted heterocyclyl, with the proviso that a carbon atom isattached to the carbonyl.

In some embodiments, R³ and R⁴ are independently selected from a groupconsisting of H, —C₁₋₉alkyl, —C₁₋₉alkylaryl and —C₁₋₉alkylheteroaryl.

In more specific embodiments, R¹ is selected from the group consistingof

In another specific embodiments, R² is selected from the groupconsisting of substituted or unsubstituted aryl,

In another specific embodiments, R³ and R⁴ are H.

Some embodiments of the present invention include compounds, salts,pharmaceutically acceptable salts or pro-drug thereof of formula (III):

In some embodiments, R¹ is selected from the group consisting ofsubstituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted carbocyclyl and substituted orunsubstituted heterocyclyl, with the proviso that a carbon atom isattached to the carbonyl.

In some embodiments, R² is selected from the group consisting ofsubstituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted carbocyclyl and substituted orunsubstituted heterocyclyl, with the proviso that a carbon atom isattached to the carbonyl.

In some embodiments, R³ and R⁴ are independently selected from a groupconsisting of H, —C₁₋₉alkyl, —C₁₋₉alkylaryl and —C₁₋₉alkylheteroaryl.

Illustrative compounds of Formulas I, II and III are shown in Table 1.

TABLE 1  1

 2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

15

16

17

18

19

20

21

Compound Preparation

The starting materials used in preparing the compounds of the inventionare known, made by known methods, or are commercially available. It willbe apparent to the skilled artisan that methods for preparing precursorsand functionality related to the compounds claimed herein are generallydescribed in the literature. The skilled artisan given the literatureand this disclosure is well equipped to prepare any of the compounds.

It is recognized that the skilled artisan in the art of organicchemistry can readily carry out manipulations without further direction,that is, it is well within the scope and practice of the skilled artisanto carry out these manipulations. These include reduction of carbonylcompounds to their corresponding alcohols, oxidations, acylations,aromatic substitutions, both electrophilic and nucleophilic,etherifications, esterification and saponification and the like. Thesemanipulations are discussed in standard texts such as Marchs AdvancedOrganic Chemistry: Reactions, Mechanisms, and Structure 6^(th) Ed., JohnWiley & Sons (2007), Carey and Sundberg, Advanced Organic Chemistry5^(th) Ed., Springer (2007), Comprehensive Organic Transformations: AGuide to Functional Group Transformations, 2^(nd) Ed., John Wiley & Sons(1999) (incorporated herein by reference in its entirety) and the like.

The skilled artisan will readily appreciate that certain reactions arebest carried out when other functionality is masked or protected in themolecule, thus avoiding any undesirable side reactions and/or increasingthe yield of the reaction. Often the skilled artisan utilizes protectinggroups to accomplish such increased yields or to avoid the undesiredreactions. These reactions are found in the literature and are also wellwithin the scope of the skilled artisan. Examples of many of thesemanipulations can be found for example in T. Greene and P. WutsProtecting Groups in Organic Synthesis, 4th Ed., John Wiley & Sons(2007), incorporated herein by reference in its entirety.

The following example schemes are provided for the guidance of thereader, and represent preferred methods for making the compoundsexemplified herein. These methods are not limiting, and it will beapparent that other routes may be employed to prepare these compounds.Such methods specifically include solid phase based chemistries,including combinatorial chemistry. The skilled artisan is thoroughlyequipped to prepare these compounds by those methods given theliterature and this disclosure. The compound numberings used in thesynthetic schemes depicted below are meant for those specific schemesonly, and should not be construed as or confused with same numberings inother sections of the application.

To further illustrate this invention, the following examples areincluded. The examples should not, of course, be construed asspecifically limiting the invention. Variations of these examples withinthe scope of the claims are within the purview of one skilled in the artand are considered to fall within the scope of the invention asdescribed, and claimed herein. The reader will recognize that theskilled artisan, armed with the present disclosure, and skill in the artis able to prepare and use the invention without exhaustive examples.

Trademarks used herein are examples only and reflect illustrativematerials used at the time of the invention. The skilled artisan willrecognize that variations in lot, manufacturing processes, and the like,are expected. Hence the examples, and the trademarks used in them arenon-limiting, and they are not intended to be limiting, but are merelyan illustration of how a skilled artisan may choose to perform one ormore of the embodiments of the invention.

¹H nuclear magnetic resonance spectra (NMR) were measured in theindicated solvents on a Bruker NMR spectrometer (Avance TM DRX300, 300MHz for 1H). Peak positions are expressed in parts per million (ppm)downfield from tetramethylsilane. The peak multiplicities are denoted asfollows, s, singlet; d, doublet; t, triplet; m, multiplet.

The following abbreviations have the indicated meanings:

Bi(OTf)₃=bismuth(III) triflate

brine=saturated aqueous sodium chloride

CDCl₃=deuterated chloroform

DMSO-d₆=deuterated dimethylsulfoxide

ESIMS=electron spray mass spectrometry

EtOAc=ethyl acetate

HCl=hydrochloric acid

MgSO₄=magnesium sulfate

NaH=sodium hydride

NMR=nuclear magnetic resonance

Ph=phenyl

K₂CO₃=potassium carbonate

rt=room temperature

TFA=trifluoroacetic acid

THF=tetrahydrofuran

TLC=thin layer chromatography

The following example schemes are provided for the guidance of thereader, and collectively represent an example method for making thecompounds provided herein. Furthermore, other methods for preparingcompounds of the invention will be readily apparent to the person ofordinary skill in the art in light of the following reaction schemes andexamples. Unless otherwise indicated, all variables are as definedabove.

General Procedures

Compounds of Formula I of the present invention can be prepared asdepicted in Scheme 1.

Scheme 1 describes a method for preparation of unsubstituted1,4-diketones derivatives (VII) by the modified Stetter reaction of aMannich base as a vinyl ketone precursor with aldehyde. The Mannich baseis formed by first reacting a methyl ketone (IV) with paraformaldehydeand dimethylamine hydrochloride to form the 3-dimethylamino-propan-1-one(V). Next, Mannich base (V) was reacted with various aldehydes (VI)under standard Stetter conditions using a thiazolium salt as thecatalyst yields unsubstituted 1,4-diketone derivatives (VII).

Compounds of Formula I of the present invention where the alpha and/orbeta positions are substituted can be prepared as depicted in Scheme 2.

Scheme 2 describes a method for preparation of substituted 1,4-diketonesderivatives (IX) by the method of Kel'in and Kulinkovich [Synthesis(1996), (3), 330-2] which is based on the application of magnesiumreagents in the cross-aldol condensation of methyl ketones with α-bromoketones. A methyl ketone (IV) is reacted with a substituted α-bromoketone (VIII) in the presence of diethylamidomagnesium bromide and acidfollowed by treatment with triethylamine to produce the desiredsubstituted 1,4-diketone derivatives (IX).

Compounds of Formula II of the present invention can be prepared asdepicted in Scheme 3.

Scheme 3 describes a method for preparation of β-diketone derivatives(VII) by a crossed Claisen condensation. A methyl ketone (IV) iscondensed with an ester (X) in the presence of sodium hydride to yieldβ-diketone derivatives (XI). The α-position can be further substitutedwith alkylbromides and base or by alkyl alcohols in the presence of aLewis acid catalyst to yield β-diketone derivatives (XII).

Compounds of Formula III of the present invention can be prepared asdepicted in Scheme 4.

Scheme 4 describes a method for preparation of γ-hydroxyketonederivatives (XIII) by the method of Xue, et al [Journal of OrganicChemistry (2006), 71(1), 215-218]. A mixture of zinc species formed from4.0 equiv of Et₂Zn, 2.0 equiv of TFA, and 4.0 equiv of CH₂I₂ efficientlyconverts β-diketones into γ-hydroxyketones. R¹ groups containingelectron-donating substituents tend to insert the cyclopropane near R²(XIII) where R¹ groups containing electron-withdrawing substituents tendto insert the cyclopropane near R¹ (XIV).

ILLUSTRATIVE COMPOUND EXAMPLES Example 1

Preparation of compound (1) is depicted below in Scheme 5.

Reagents and conditions: a) Ethanol, HCHO, HCl, refluxed, overnight; b)Dioxane, PhCHO, 3-Ethyl-5-(2-hydroxyethyl)-4-methylthi azolium bromide,95° C., overnight.

Step a

A solution of 1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)ethanone (XV) (11mmol), dimethylamine hydrochloride (14 mmol), paraformaldehyde (16 mmol)and 12 N HCl (2 drops) in ethanol (5 mL) was refluxed overnight. Thesolution was cooled to room temperature and the ethanol was evaporatedunder vacuum. The residue was treated with ethyl acetate, heatedslightly and sonicated to disperse into fine particles. The solids werefiltered and dried at room temperature to produce1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-(dimethylamino)propan-1-one(XVI) as a white solid, (82% yield), ¹H NMR (CDCl₃, 400 MHz): δ ppm 2.77(s, 6H), 3.41 (m, 2H), 3.56 (m, 2H), 4.25 (m, 4H), 6.85 (m, 1H), 7.45(m, 2H).

Step b

Triethylamine (3.61 mmol) and benzaldehyde (4.3 mmol) in dry dioxane (10mL) was added to a solution of1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-(dimethylamino)propan-1-one(XVI) (5.4 mmol) and 3-ethyl-5-(2-hydroxyethyl)-4-methylthiazoliumbromide (0.43 mmol) in dioxane heated at 95° C. under nitrogen. Thesolution was further heated overnight at 95° C. The solution was cooledand excess solvent was evaporated under vacuum. The residue waspartitioned between CH₂Cl₂ and water. The organic phase was dried overMgSO₄, filtered and concentrated. The residue was purified by flashchromatography eluting with ethyl acetate in hexane gradient to yield1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4-phenylbutane-1,4-dione 1 as awhite solid (12% yield). ¹H NMR (DMSO-d₆, 400 MHz): δ ppm 3.33-3.37 (m,4H), 4.29 (m, 2H), 4.34 (m, 2H), 6.98 (m, 1H), 7.48 (m, 1H), 7.55 (m,3H), 7.64 (m, 1H), 8.00-8.02 (m, 2H); ESIMS found C₁₈H₁₆O₄ m/z 297(M+H).

The following compounds was prepared in accordance with the proceduredescribed in the above Example 1.

1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4-(pyridin-2-yl)butane-1,4-dione2

White solid. ¹H NMR (CDCl₃, 400 MHz): δ ppm 3.37 (m, 2H), 3.40 (m, 2H),4.31 (m, 4H), 6.92 (m, 1H), 7.47 (m, 2H), 7.56 (m, 1H), 7.83 (m, 1H),8.03 (m, 1H), 8.71 (m, 1H); ESIMS found C₁₇H₁₅NO₄ m/z 298 (M+H).

1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4-(thiophen-2-yl)butane-1,4-dione3

Yellow solid. ¹H NMR (DMSO-d₆, 400 MHz): δ ppm 4.29 (m, 2H), 4.34 (m,2H), 6.97 (m, 1H), 7.25 (m, 1H), 7.47 (m, 1H), 7.52 (m, 1H), 8.00-8.03(m, 2H); ESIMS found C₁₆H₁₄O₄S m/z 303 (M+H).

1-(pyridin-2-yl)-4-(thiophen-2-yl)butane-1,4-dione 5

White solid. ¹H NMR (DMSO-d₆, 400 MHz): δ ppm 3.40 (m, 2H), 3.56 (m,2H), 7.27 (dd, J=4.8, 3.8 Hz, 1H), 7.70 (m, 1H), 7.95 (d, J=7.8 Hz, 1H),8.00-8.05 (m, 3H), 8.76 (d, J=4.3 Hz, 1H); ESIMS found C₁₃H₁₁NO₂S m/z246 (M+H).

1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4-(4-fluorophenyl)butane-1,4-dione7

Off white solid. ¹H NMR (CDCl₃, 400 MHz): δ ppm 3.40 (s, 4H), 4.30 (m,4H), 6.92 (m, 1H), 7.16 (m, 2H), 7.57 (m, 2H), 8.04 (m, 2H); ESIMS foundC₁₈H₁₅FO₄ m/z 315 (M+H).

1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4-(4-methoxyphenyl)butane-1,4-dione10

White solid (19% yield). ¹H NMR (DMSO-d₆, 400 MHz): δ ppm 3.85 (s, 3H),4.30 (m, 2H), 4.34 (m, 2H), 6.99 (d, J=8.3 Hz, 1H), 7.06 (d, J=8.8 Hz,2H), 7.48 (d, J=2.0 Hz, 1H), 7.54 (dd, J=8.3, 2.0 Hz, 1H), 7.98 (d,J=8.8 Hz, 2H); ESIMS found C₁₉H₁₈O₅ m/z 327 (M+H).

1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4-(4-((2-methoxyethoxy)methoxy)phenyl)butane-1,4-dione 11

Off white solid. ¹H NMR (DMSO-d₆, 400 MHz): δ ppm 3.33 (m, 4H), 3.46 (m,2H), 3.75 (m, 2H), 4.30 (m, 2H), 4.34 (m, 2H), 5.36 (s, 2H), 6.99 (d,J=8.3 Hz, 1H), 7.14 (d, J=8.8 Hz, 2H), 7.48 (d, J=2.0 Hz, 1H), 7.55 (dd,J=8.5, 2.0 Hz, 1H), 7.98 (d, J=8.8 Hz, 2H); ESIMS found C₂₂H₂₄O₇ m/z 401(M+H).

1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4-(3-((2-methoxyethoxy)methoxy)phenyl)butane-1,4-dione 12

White solid. ¹H NMR (DMSO-d₆, 400 MHz): δ ppm 3.21 (s, 3H), 3.35 (m,4H), 3.48 (m, 2H), 3.75 (m, 2H), 4.30 (m, 2H), 4.33 (m, 2H), 5.32 (s,2H), 6.99 (d, J=8.3 Hz, 1H), 7.31 (dd, J=8.2, 2.0 Hz, 1H), 7.49 (m, 2H),7.55 (dd, J=8.5, 2.0 Hz, 1H), 7.58 (m, 1H), 7.67 (d, J=7.8 Hz, 1H);ESIMS found C₂₂H₂₄O₇ m/z 401 (M+H).

1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4-(2-((2-methoxyethoxy)methoxy)phenyl)butane-1,4-dione 13

Viscous oil (14% Yield). ¹H NMR (DMSO-d₆, 400 MHz): δ ppm 3.21 (s, 3H),3.25-3.29 (m, 4H), 3.46-3.48 (m, 2H), 3.79 (m, 2H), 4.29 (m, 2H), 4.33(m, 2H), 5.39 (s, 2H), 6.98 (d, J=8.6 Hz, 1H), 7.10 (m, 1H), 7.25 (dd,J=8.3 Hz, 1H), 7.47 (m, 1H), 7.49-7.54 (m, 2H), 7.58 (dd, J=7.7, 1.6 Hz,1H); ESIMS found C₂₂H₂₄O₇ m/z 401 (M+H).

1,4-bis(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)butane-1,4-dione 14

Off white solid. ¹H NMR (CDCl₃, 400 MHz): δ ppm 3.35 (s, 4H), 4.30 (m,8H), 6.92 (m, 2H), 7.57 (m, 4H); ESIMS found C₂₀H₁₈O₆ m/z 355 (M+H).

Example 2

Preparation of compound (4) is depicted below in Scheme 6.

Reagents and conditions: a) CH₂Cl₂, TFA, rt, overnight.

Step a

Neat TFA (0.5 mL) was added to a solution of1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4-(4-((2-methoxyethoxy)methoxy)phenyl)butane-1,4-dione11 (0.35 mmol) in CH₂Cl₂ (5 mL) stirred at room temperature. Thesolution was further stirred overnight at room temperature. Thevolatiles were evaporated under vacuum. The residue was purified byflash chromatography over silica gel eluting with 1% methanol in CH₂Cl₂to get1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4-(4-hydroxyphenyl)butane-1,4-dione4 as an off white solid (21% yield). ¹H NMR (CDCl₃, 400 MHz): δ ppm 3.37(m, 4H), 4.29 (m, 2H), 4.33 (m, 2H), 5.76 (s, 1H), 6.87 (d, J=8.8 Hz,2H), 6.93 (m, 1H), 7.60 (m, 2H), 7.94 (d, J=8.8 Hz, 2H); ESIMS foundC₁₈H₁₆O₅ m/z 313 (M+H).

The following compounds was prepared in accordance with the proceduredescribed in the above Example 2.

1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4-(3-hydroxyphenyl)butane-1,4-dione6

Off white solid (27% yield). ¹H NMR (DMSO-d₆, 400 MHz): δ ppm 3.27 (m,4H), 4.30 (m, 2H), 4.34 (m, 2H), 6.99 (d, J=8.8 Hz, 1H), 7.03 (d, J=8.8Hz, 1H), 7.36 (m, 2H), 7.45 (m, 2H), 7.54 (d, J=8.8 Hz, 1H).

1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4-(2-hydroxyphenyl)butane-1,4-dione8

Off white solid (62% yield). ¹H NMR (DMSO-d₆, 400 MHz): δ ppm 3.43 (m,4H), 4.00 (m, 4H), 4.34 (m, 2H), 6.96 (m, 3H), 7.48-7.56 (m, 3H), 7.97(m, 1H).

Example 3

Preparation of compound (9) is depicted below in Scheme 7.

Reagents and conditions: a) i) Et₂NMgBr.Et₂O, Toluene, 0° C., 3 h ii)H₂SO₄, H₂O, 0° C.-rt, iii) Et₃N, rt.

Step a

In a dried 3 neck flask fitted with magnetic stirrer and condenser wasplaced metal magnesium (12 mmol) and ether (1.8 mL). Neat bromoethane(2.5 mmol) was added via syringe and the reaction was startedimmediately. A solution of bromoethane (10.5 mmol) in toluene (30 mL)was added to the solution slowly. After completion of the addition, thesolution was stirred at room temperature for 30 min under nitrogenbefore adding neat diethylamine (24 mmol). The solution was furtherstirred at room temperature for 15 min. The solution was cooled to 0° C.and a mixture of 1-(pyridin-2-yl)ethanone (XVIII) (12 mmol) and2-bromoisobutyrophenone (XVII) (13 mmol) was added to the solution. Thesolution was further stirred for 3 h at 0° C. under nitrogen. Aqueous 5%H₂SO₄ (20 mL) was added to the solution and the solution was warmed toroom temperatue. The organic layer was separated, dried over MgSO₄ andfiltered. The organic layer was then treated with Et₃N (10 mmol) andallowed to stir overnight at room temperature. The solution was thenwashed with water, dried over MgSO₄, filtered and concentrated. Theresidue was purified by flash chromatography eluting with 1-5% EtOAc inhexane gradient to give2,2-dimethyl-1-phenyl-4-(pyridin-2-yl)butane-1,4-dione 9 as a colorlessviscous oil (11% yield). ¹H NMR (CDCl₃, 400 MHz): δ ppm 1.47 (s, 6H),3.80 (s, 2H), 7.34-7.50 (m, 4H), 7.65-7.75 (m, 2H), 7.80 (m, 1H), 7.94(m, 1H), 8.67 (m, 1H); ESIMS found C₁₇H₁₇NO₂ m/z 268 (M+H).

Example 4

Preparation of compound (15) is depicted below in Scheme 8.

Reagents and conditions: a) THF, NaH, rt-reflux, overnight.

Step a

A solution of 1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)ethanone (XV) (1eq) in THF was added slowly to a suspension of NaH (1.5 eq) in THFstirred under nitrogen at room temperature. The solution was furtherallowed to stir at room temperature until the evolution of gas wasceased. Ethyl picolinate (XIX) (1.1 eq) was added to the solution andrefluxed overnight under nitrogen. The solution was cooled, poured intoice water and extracted with ethyl acetate. The organic layer was driedover MgSO₄, filtered and concentrated under vacuum. The crude productwas purified by column chromatography over silica gel to produce1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-(pyridin-2-yl)propane-1,3-dione15 as a yellow solid (71% yield), ¹H NMR (CDCl₃, 400 MHz): δ ppm4.13-4.43 (m, 4H), 6.94 (d, J=8.31 Hz, 1H), 7.42 (m, 1H), 7.44 (m, 1H)7.59 (m, 2H), 7.85 (m, 1H), 8.14 (d, J=7.81, 1H), 8.65 (m, 1H); ESIMSfound C₁₆H₁₃NO₄ m/z 284 (M+H).

The following compounds was prepared in accordance with the proceduredescribed in the above Example 4.

1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-(thiophen-2-yl)propane-1,3-dione16

Yellow solid. ¹H NMR (DMSO-d₆, 400 MHz): δ ppm 4.34 (m, 4H), 6.98 (m,1H), 7.15 (br. s, 1H), 7.59 (m, 1H), 7.64 (m, 2H), 8.01 (m, 1H), 8.28(m, 1H); ESIMS found C₁₅H₁₂O₄S m/z 289 (M+H).

Example 5

Preparation of compounds (17) and (18) are depicted below in Scheme 9.

Reagents and conditions: a) DMSO, bromoethane, K₂CO₃, rt, overnight.

Step a

Bromoethane (0.87 mmol) was added slowly to a solution1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-phenylpropane-1,3-dione (XX)(0.39 mmol) and K₂CO₃ (1.58 mmol) in DMSO (4 mL) stirred at roomtemperature under nitrogen. The solution was further stirred overnightat room temperature under nitrogen. The solution was poured into amixture of water and ether. The etheric layer was separated, washed withbrine, dried over MgSO₄, filtered and concentrated. The residue waspurified by column chromatography to get1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-ethyl-3-phenylpropane-1,3-dione17 as colorless viscous oil (35% yield). ¹H NMR (DMSO-d₆, 400 MHz): δppm 0.93 (t, J=7.4 Hz, 3H), 1.93 (m, 2H), 4.28 (m, 2H), 4.33 (m, 2H),5.60 (t, J=6.5 Hz, 1H), 6.99 (d, J=8.6 Hz, 1H), 7.51-7.55 (m, 4H), 7.66(m, 1H), 7.98 (d, J=7.3 Hz, 2H); ESIMS found C₁₉H₁₈O₄ m/z 311 (M+H) and1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2,2-diethyl-3-phenylpropane-1,3-dione18 as a white solids. ESIMS found C₂₁H₂₂₀₄ m/z 339 (M+H).

Example 6

Preparation of compound (19) is depicted below in Scheme 10.

Reagents and conditions: a) CH₃NO₂, Bi(OTf)₃, 100° C., 2 h

Step a

A solution of naphthalen-1-ylmethanol (XXII) (0.75 mmol) in CH₃NO₂ (1mL) was added slowly over a period of 45 min to a solution ofdibenzoylmethane (XXI) (2.27 mmol) and Bi(OTf)₂ (0.008 mmol) in CH₃NO₂heated at 100° C. The solution was further stirred at 100° C. for 2 h.The solution was cooled and the solvent was removed under vacuum. Theresidue was purified by column chromatography to produce2-(naphthalen-1-ylmethyl)-1,3-diphenylpropane-1,3-dione 19 as a yellowsolid (75% yield). ¹H NMR (DMSO-d₆, 400 MHz): δ ppm 3.75 (d, J=7 Hz,2H), 6.23 (t, J=7 Hz, 1H), 7.24-7.38 (m, 6H), 7.50-7.55 (m, 4H), 7.57(m, 1H), 7.81-7.87 (m, 5H), 8.14 (m, 1H); ESIMS found C₂₆H₂₀O₂ m/z 365(M+H).

Administration and Pharmaceutical Compositions

Some embodiments include pharmaceutical compositions comprising: (a) asafe and therapeutically effective amount of a compound according toFormulas I, II or III, or its corresponding enantiomer, diastereoisomeror tautomer, or pharmaceutically acceptable salt; and (b) apharmaceutically acceptable carrier.

Administration of the compounds disclosed herein or the pharmaceuticallyacceptable salts thereof can be via any of the accepted modes ofadministration for agents that serve similar utilities including, butnot limited to, orally, subcutaneously, intravenously, intranasally,topically, transdermally, intraperitoneally, intramuscularly,intrapulmonarilly, vaginally, rectally, or intraocularly. Oral andparenteral administrations are customary in treating the indications.

Compounds of the invention intended for pharmaceutical use may beadministered as crystalline or amorphous products. Pharmaceuticallyacceptable compositions include solid, semi-solid, liquid and aerosoldosage forms, such as, e.g., tablets, capsules, powders, liquids,suspensions, suppositories, aerosols or the like. They may be obtained,for example, as films by methods such as precipitation, crystallization,freeze drying, spray drying, or evaporative drying. Microwave or radiofrequency drying may be used for this purpose. The compounds can also beadministered in sustained or controlled release dosage forms, includingdepot injections, osmotic pumps, pills, transdermal (includingelectrotransport) patches, and the like, for prolonged and/or timed,pulsed administration at a predetermined rate. Preferably, thecompositions are provided in unit dosage forms suitable for singleadministration of a precise dose.

The compounds can be administered either alone or more typically incombination with a conventional pharmaceutical carrier, excipient or thelike. The term “excipient” is used herein to describe any ingredientother than the compound(s) of the invention. Pharmaceutically acceptableexcipients include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, self-emulsifying drug delivery systems(SEDDS) such as d-α-tocopherol polyethylene glycol 1000 succinate,surfactants used in pharmaceutical dosage forms such as Tweens or othersimilar polymeric delivery matrices, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate,sodium-chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethyl cellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, and wool fat.Cyclodextrins such as α-, β, and γ-cyclodextrin, or chemically modifiedderivatives such as hydroxyalkylcyclodextrins, including 2- and3-hydroxypropyl-b-cyclodextrins, or other solubilized derivatives canalso be advantageously used to enhance delivery of compounds of theformulae described herein. Dosage forms or compositions containing acompound as described herein in the range of 0.005% to 100% with thebalance made up from non-toxic carrier may be prepared. The contemplatedcompositions may contain 0.001%-100% active ingredient, in oneembodiment 0.1-95%, in another embodiment 75-85%. Actual methods ofpreparing such dosage forms are known, or will be apparent, to thoseskilled in this art; for example, see Remington: The Science andPractice of Pharmacy, 21st Edition (Lippincott Williams & Wilkins.2005).

In one preferred embodiment, the compositions will take the form of aunit dosage form such as a pill or tablet and thus the composition maycontain, along with the active ingredient, a diluent such as lactose,sucrose, dicalcium phosphate, or the like; a lubricant such as magnesiumstearate or the like; and a binder such as starch, gum acacia,polyvinylpyrrolidine, gelatin, cellulose, cellulose derivatives or thelike. In another solid dosage form, a powder, marume, solution orsuspension (e.g., in propylene carbonate, vegetable oils ortriglycerides) is encapsulated in a gelatin capsule. Unit dosage formsin which the two active ingredients are physically separated are alsocontemplated; e.g., capsules with granules of each drug; two-layertablets; two-compartment gel caps, etc.

In another preferred embodiment, compositions described herein are usedas a drug-eluting coatings for a medical device including, but notlimited to temporary or permanent implants, sponge, polymer, or gel.

The implant according to an embodiment of the invention is an orthopedicimplant including, but not limited to (i) a hip joint, (ii) screws,cannulated screws, nails, meshes, cages, wires, pins, intramedullarynails, rods, posts, anchors, and plates intended to join or attach bonefragments, pieces, or parts with each other, (iii) external skeletalfixators such as monolateral, multiplanar or hybrid fixators, (iv)implants intended for treatment of degenerative instabilities,fractures, tumors, and deformities in respect of the spine, (v)cranio-maxillofacial implants intended for treatment of fractures,reconstruction, and correction of deformities, of mandible, mid-face, orskull, (vi) surgical stents, collagen stents, intramedullary bonestents, (vii) anterior cruciate ligament (ACL) and posterior cruciateligament (PCL) Reconstruction Systems, (viii) dental implants.

In some instances, a compound according to Formulas I, II or III isadministered in combination with one or more therapeutic agents, e.g.,therapeutic agents useful in the treatment of bone disorders orconditions described herein. For example, certain second therapeuticagents can promote tissue growth or infiltration, such as growthfactors. Exemplary growth factors for this purpose include, withoutlimitation, epidermal growth factor (EGF), vascular endothelial growthfactor (VEGF), fibroblast growth factor (FGF), platelet-derived growthfactor (PDGF), transforming growth factors (TGFs), parathyroid hormone(PTH), leukemia inhibitory factor (LIF), and insulin-like growth factors(IGFs). Other second therapeutic agents can promote bone growth, such asbone morphogenetic proteins (U.S. Pat. No. 4,761,471; PCT Pub. WO90/11366), osteogenin (Sampath, et al., Proc. Natl. Acad. Sci. USA(1987), 84(20), 7109-7113), NaF (Tencer, et al., Journal of BiomedicalMaterials Research (1989), 23(6), 571-589), Peptide sequences such asIKVAV may be added to attach nerves and have those nerves expressneuritis (Tashiro, et al., The Journal of Biological Chemistry (1989),264(27), 16174-16182).

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, etc. an active compound as definedabove and optional pharmaceutical adjuvants in a carrier (e.g., water,saline, aqueous dextrose, glycerol, glycols, ethanol or the like) toform a solution or suspension. If desired, the pharmaceuticalcomposition can also contain minor amounts of nontoxic auxiliarysubstances such as wetting agents, emulsifying agents, solubilizingagents, pH buffering agents and the like (e.g., sodium acetate, sodiumcitrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamineacetate, triethanolamine oleate, and the like). Injectables can beprepared in conventional forms, either as liquid solutions orsuspensions, as emulsions, or in solid forms suitable for dissolution orsuspension in liquid prior to injection. The percentage of activecompound contained in such parenteral compositions is highly dependenton the specific nature thereof, as well as the activity of the compoundand the needs of the subject. However, percentages of active ingredientof 0.01% to 10% in solution are employable, and will be higher if thecomposition is a solid, which will be subsequently diluted to the abovepercentages. In some embodiments, the composition will comprise 0.2-2%of the active agent in solution.

It is to be noted that concentrations and dosage values may also varywith the severity of the condition to be alleviated. It is to be furtherunderstood that for any particular patient, specific dosage regimensshould be adjusted over time according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the compositions, and that the concentration rangesset forth herein are exemplary only and are not intended to limit thescope or practice of the claimed compositions.

Solid compositions can be provided in various different types of dosageforms, depending on the physicochemical properties of the drug, thedesired dissolution rate, cost considerations, and other criteria. Inone of the embodiments, the solid composition is a single unit. Thisimplies that one unit dose of the drug is comprised in a single,physically shaped solid form or article. In other words, the solidcomposition is coherent, which is in contrast to a multiple unit dosageform, in which the units are incoherent.

Examples of single units which may be used as dosage forms for the solidcomposition include tablets, such as compressed tablets, film-likeunits, foil-like units, wafers, lyophilized matrix units, and the like.In a preferred embodiment, the solid composition is a highly porouslyophilized form. Such lyophilizates, sometimes also called wafers orlyophilized tablets, are particularly useful for their rapiddisintegration, which also enables the rapid dissolution of the activecompound.

On the other hand, for some applications the solid composition may alsobe formed as a multiple unit dosage form as defined above. Examples ofmultiple units are powders, granules, microparticles, pellets, beads,lyophilized powders, and the like. In one embodiment, the solidcomposition is a lyophilized powder. Such a dispersed lyophilized systemcomprises a multitude of powder particles, and due to the lyophilizationprocess used in the formation of the powder, each particle has anirregular, porous microstructure through which the powder is capable ofabsorbing water very rapidly, resulting in quick dissolution.

Another type of multiparticulate system which is also capable ofachieving rapid drug dissolution is that of powders, granules, orpellets from water-soluble excipients which are coated with the drug, sothat the drug is located at the outer surface of the individualparticles. In this type of system, the water-soluble low molecularweight excipient is useful for preparing the cores of such coatedparticles, which can be subsequently coated with a coating compositioncomprising the drug and, preferably, one or more additional excipients,such as a binder, a pore former, a saccharide, a sugar alcohol, afilm-forming polymer, a plasticizer, or other excipients used inpharmaceutical coating compositions.

Also provided herein are kits. Typically, a kit includes one or morecompounds or compositions as described herein. In certain embodiments, akit can include one or more delivery systems, e.g., for delivering oradministering a compound as provided above, and directions for use ofthe kit (e.g., instructions for treating a patient). In anotherembodiment, the kit can include a compound or composition as describedherein and a label that indicates that the contents are to beadministered to a patient with cancer. In another embodiment, the kitcan include a compound or composition as described herein and a labelthat indicates that the contents are to be administered to a patientwith one or more of osteoporosis and osteoarthropathy; osteogenesisimperfecta, bone defects, bone fractures, periodontal disease,otosclerosis, wound healing, craniofacial defects, oncolytic bonedisease, traumatic brain injuries related to the differentiation anddevelopment of the central nervous system, comprising Parkinson'sdisease, strokes, ischemic cerebral disease, epilepsy, Alzheimer'sdisease, depression, bipolar disorder, schizophrenia; eye diseases suchas age related macular degeneration, diabetic macular edema, familialexudative vitreoretinopathy or retinitis pigmentosa and diseases relatedto differentiation and growth of stem cell, comprising hair loss,hematopoiesis related diseases, tissue regeneration related diseases andother diseases associated with abnormalities in development, stem celldifferentiation and cell proliferation.

The actual dose of the active compounds of the present invention dependson the specific compound, and on the condition to be treated; theselection of the appropriate dose is well within the knowledge of theskilled artisan.

Methods of Treatment

The compounds and compositions provided herein can be used as activatorsof one or more members of the Wnt pathway, including one or more Wntproteins, and thus can be used to treat a variety of disorders anddiseases in which aberrant Wnt signaling is implicated, such asosteoporosis and osteoarthropathy; osteogenesis imperfecta, bonedefects, bone fractures, periodontal disease, otosclerosis, woundhealing, craniofacial defects, oncolytic bone disease, traumatic braininjuries related to the differentiation and development of the centralnervous system, comprising Parkinson's disease, strokes, ischemiccerebral disease, epilepsy, Alzheimer's disease, depression, bipolardisorder, schizophrenia; eye diseases such as age related maculardegeneration, diabetic macular edema, familial exudativevitreoretinopathy or retinitis pigmentosa and diseases related todifferentiation and growth of stem cell, comprising hair loss,hematopoiesis related diseases, tissue regeneration related diseases andother diseases associated with abnormalities in development, stem celldifferentiation and cell proliferation.

With respect to hair loss, the canonical Wnt/β-catenin signaling pathwayis known to regulate hair follicle development and regeneration. In theepidermis, hair follicle development is initiated when mesenchymal cellspopulate the skin. During this process, signals emanating from thedermis induce epithelium thickening, elongation of the epithelial cells,and the formation of placodes containing Wnt-responsive cells. Inresponse, placodes signal dermal cells to condense, thereby forming thedermal papilla component of the hair follicle, which is also responsiveto Wnt signaling. Wnt3a is secreted from hair epithelium and acts in anautocrine and paracrine fashion, and it has been demonstrated thatWnt-3a maintains anagen gene expression in dermal papilla cells andmediates hair-inductive activity in an organ culture. ThisWnt-3a-mediated hair growth might depend on the canonical Wnt/β-cateninsignaling pathway because deletion of 3-catenin or the Lef1 generesulted in hair loss in mice. Accordingly, the compounds andcompositions described herein may be used topically to treat hair lossby modulation of the Wnt/β-catenin signaling pathway.

With respect to neurodegenerative diseases, Wnt/β-catenin signaltransduction system plays a crucial role in the differentiation anddevelopment of nerve cells for the central nervous system. Particularly,it is found that Wnt/β-catenin signaling is related to diseasesresulting from the abnormality of nerve cells.

More particularly in Alzheimer's disease, studies indicate that asustained loss of Wnt signaling function may be involved in theAβ-dependent neurodegeneration observed in Alzheimer's brain.Consequently, the compounds and compositions described herein may beused to reactivate lost Wnt signaling function involved inneurodegeneration.

Other neurodegenerative diseases can also be treated with the compoundsand compositions described herein.

More particularly, neurodegenerative diseases that may be treated by thecompound, compositions and methods described herein include, but are notlimited to, the following:

Parkinson's disease, schizophrenia, Huntington's disease, amyotrophiclateral sclerosis (Lou Gehrig's disease), primary lateral sclerosis(PLS), progressive muscular atrophy (PMA), bipolar disorder, depression,strokes, ischemic cerebral disease, epilepsy, brain damage andspinocerebellar ataxia type 1 (SCA1).

With respect to eye diseases, Wnt/β-catenin signal transduction systemregulates the maintenance of a retinal progenitor population in theciliary marginal zone (CMZ), and thus function as a putative stem cellfactor in the retina. Particularly, it is found that Wnt/β cateninpathways mediate a process of retinal repair and that application of Wntactivators may promote retinal neuron regeneration. In the setting ofinjury, Wnts may serve a protective role. It has been recently shownthat Wnt3a protects photoreceptors. The results of this study may wellbe interpreted as an upregulation of self-renewal of stem cells in thesetting of injury.

Accordingly, the compounds and compositions described herein may be usedto enhance replacement of lost neurons caused by disease and protectphotoreceptors during injury by modulation of the Wnt/β-cateninsignaling pathway.

Other eye diseases can also be treated with the compounds andcompositions described herein.

More particularly, eye diseases that may be treated by the compound,compositions and methods described herein include, but are not limitedto, the following:

Age related macular degeneration, diabetic macular edema, familialexudative vitreoretinopathy and retinitis pigmentosa.

With respect to diseases associated with differentiation and growth ofstem cell, Wnt/β-catenin signaling is critical in the self-renewal ofstem cells in many different tissues, including the skin, intestine,brain and blood. Therefore, the compounds and compositions describedherein may be used to treat disorders and diseases related toabnormalities in development.

Evaluation of Biological Activity

The biological activity of the compounds described herein can be testedusing any suitable assay known to those of skill in the art, e.g., WO2001/053268 or WO 2005/009997. For example, the activity of a compoundmay be tested using one or more of the test methods outlined below.

Example 7

Compounds that enhance the Wnt activity, or Activators, were assayed asfollows. Reporter cell lines were generated by stably transducing cellsof cancer cell lines (e.g., colon cancer) with a lentiviral constructthat include a wnt-responsive promoter driving expression of the fireflyluciferase gene.

Lentiviral constructs were made in which the SP5 promoter, a promoterhaving eight TCF/LEF binding sites derived from the SP5 promoter, islinked upstream of the firefly luciferase gene. The lentiviralconstructs can also include a hygromycin resistance gene as a selectablemarker. The SP5 promoter construct were used to transduce SW480 cells, acolon cancer cell line having a mutated APC gene that generates atruncated APC protein, leading to de-regulated accumulation ofβ-catenin.

Cultured SW480 cells bearing a reporter construct can be distributed atapproximately 10,000 cells per well into 384 or 96 well multiwellplates. Compounds from a small molecule compound library can then beadded to the wells in half-log dilutions using three or ten micromolartop concentration. A series of control wells for each cell type receivedonly buffer and compound solvent DMSO. Twenty-four hours after theaddition of compound, reporter activity for luciferase can be assayed,for example, by addition of the BrightGlo luminescence reagent (Promega)and the Victor3 plate reader (Perkin Elmer). Readings are normalized toDMSO only treated cells, and any activities above DMSO are consideredactivation. Compounds are considered activators if reporter activitiesare 2× fold or greater than DMSO. EC₅₀ is the concentration at halfmaximal activation. Table 2 shows the activity of selected activators.

TABLE 2 Compound Wnt activation, IC₅₀ 1 0.028-0.029 μM 2 0.013 μM 30.036-0.041 μM 4 0.61-1.0 μM 5 0.64-1.9 μM 6 2.1 μM 7 0.096-0.27 μM 80.083 μM 9 >10 μM 10 >10 μM 11 >10 μM 12 >10 μM 14 >10 μM 15 0.68-2.1 μM16 2.57 μM 17 >10 μM 18 6.0 μM 19 2.8-5.0 μM

The term “comprising” as used herein is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps.

1. (canceled)
 2. A compound or pharmaceutically acceptable salt orprodrug thereof having the structure of Formula II:

wherein R¹ is selected from the group consisting of substituted orunsubstituted heteroaryl and substituted or unsubstituted heterocyclyl,with the proviso that a carbon atom is attached to the carbonyl; R² isselected from the group consisting of substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedcarbocyclyl and substituted or unsubstituted heterocyclyl, with theproviso that a carbon atom is attached to the carbonyl; and R³ and R⁴are independently selected from a group consisting of H, —C₁₋₉alkyl,—C₁₋₉alkylaryl and —C₁₋₉alkylheteroaryl.
 3. The compound of claim 2,wherein R¹ is selected from the group consisting of


4. The compound of claim 3, wherein R² is selected from the groupconsisting of substituted or unsubstituted aryl,


5. The compound of claim 4, wherein R³ and R⁴ are H.
 6. The compound ofclaim 2, having a structure selected from the group consisting of:

or pharmaceutically acceptable salt thereof.